Description
CHM/BIOS-372
Elements of Biochemistry II
Final Exam Study Guide
5/1/2017
Introduction to Metabolism
Course Objectives
∙ Develop an understanding of major metabolic pathway details ∙ Learn the major ways in which metabolic pathways are regulated ∙ Focus on how metabolic pathways are involved in specific aspects of physiology
Chemical Reactions in Your Bodies – Fig. 16.1
∙ Complicated diagram shows how reaction pathways interrelate ∙ There is a fair amount of coordinate control
∙ Ex: “Epigenometric reprogramming…” etc. shows that this material is ongoing and relevant because this article was posted Jan 17, 2017 Section Goals
∙ Introduce concepts and terminology we will encounter
If you want to learn more check out What was the nullification crisis in 1832-1837?
regularly in our study of metabolism
∙ To begin our examination of thermodynamics as a
major player in control of metabolism
Metabolic Pathways/Terms
A. Terms and Concepts – Fig. 16.2
a. Phototrophs – plants etc. that get energy
from the sun
b. Chemotrophs – rest of life etc. that gets
energy from chemical molecules (ingestion)
c. Anabolism – adding up
d. Catabolism – breaking apart Don't forget about the age old question of Who is vladimir lenin?
e. Fig. 16.2 – Biosynthesis is when the body takes simple
molecules and uses them to make complex metabolites (a
substance formed in or necessary for metabolism)
B. Ways of Grouping Reactions – Fig. 16.3
a. Pathways for specific goals in metabolism
b. Fig. 16.3 – shows various pathways of proteins, carbohydrates, and lipids through our body and how they relate to glycolysis, pyruvate, acetyl-CoA, the citric acid cycle, and oxidative
phosphorylation
C. Control Mechanisms for Metabolic Pathways
a. Regulation of pathways – diet helps regulate pathways
b. Irreversibility of Pathways – need alternative pathways for back-reactions
c. Committed Steps – makes decision about resources in our bodies (Ex: Incoming AA, should we break them down or keep them whole?)
d. Isolation by Location
i. Ex: Livers carry out specific reactions as well as other tissues having unique functions
ii. Ex: Same thing with cells – cytoplasm and membrane surfaces deal with the catabolism of fatty acids while mitochondria degrades them If you want to learn more check out What are the economic variables of the development of russia?
D. Chemical Logic – Fig. 16.5
a. Nucleophile to electrophile
i. ^Fig. 16.5 shows examples of nucleophilic forms
ii. Nucleophile – a chemical species that donates an electron pair to an If you want to learn more check out What model shows the flow of memory between 3 different types of memory?
electrophile to form a chemical bond in relation to a reaction – All molecules or ions with a free pair of electrons or at least one pi bond can act as nucleophiles – Because nucleophiles donate electrons, they are by definition Lewis Bases
iii. Electrophile – a reagent attracted to
electrons – They are positive or neutral
species having vacant orbitals that are
attracted to an electron rich center – It
participates in a chemical reaction by We also discuss several other topics like What is the meaning of solar flux?
accepting an electron pair in order to bind
to a nucleophile
b. Group Transfer Reactions Include – Fig. 16.7
i. Larger structures are typically transferred
in anabolic and catabolic reactions
ii. Common types are acyl, phosphoryl, and
glycosyl group transfers
iii. Ex: Fig. 16.7 – glucose and ATP combine
with a phosphate group to make a
trigonal bipyramid intermediate and then
form Glucose-6-phosphate and ADP (the
phosphate group is a group transfer)
c. Oxidation-Reduction Reactions
i. Oxidation/Catabolic – breaking down - reactions are accompanied by reduction of an electron carrier
a. H3C(CH2)3OH → H3C(CH2)2CH=O (+ CO2 as a byproduct)
ii. Reduction/Anabolic – making up - reactions need lots of electrons from electron carriers
a. H3CCOO- → H3C(CH2)nCOO
d. Eliminations, Isomerizations, Rearrangements
i. Elimination of water Don't forget about the age old question of What is the meaning of slum?
ii. Keto- to Enol- conversions
iii. Rearrangements are less common
e. Make and Break Carbon-Carbon Bonds. All Types of Reactions
i. Ex: Breakdown of glucose to carbon dioxide involves separating all 6 carbons from each other to produce 6 CO2
E. Techniques for Studying Metabolism - Fig. 16.17
a. Metabolic Inhibitors and a little intuition
b. Genetic Defects – phenylketonuria is a well-known example in which patients are unable to process pheylalanine
c. Genetic manipulation to generate
blocks in a pathway – pick and
choose through careful
consideration
d. Isotopes (one example in next
paragraph)
e. Isolated Organelles – Ex: Can do a
lot with just a mitochondria
f. Metabolomics, etc.
g. Fig. 16.17 – using radioisotopes to
figure out how heme is made using
radioactive isotopes to find the
individual building blocks
Thermodynamics in Biological Systems
A. Basic Termodynamics
a. Biological conditions!!
Remember that the body is in
aqueous solution and
physiological pH is 7
b. Find formulas necessary for thermodynamic calculations in chapter 3, p. 58 in textbook (same as above tho)
B. Steady State or Death!! – Fig. 16.31
a. Equilibrium is in a closed system, while biological systems are open!
b. Therefore, biological systems are never in equilibrium, they are in steady state c. Ex: Photos of a river will look the same from one day to another but we know the water molecules are not the same
d. Ex: Same with pathways in our bodies –
individual molecules are changing but levels stay
relatively the same
C. Coupling Reactions – Fig. 16.23
a. The ending dG value of -16.7kJmol-1 is still
negative (favorable) even though the endergonic
half reaction requires energy and is unfavorable
b. Enzyme Coupled Reactions Fig. 16.32
i. Have a test tube with aqueous buffer
ii. Energy barrier for overall reaction is too
high so we know that it could possibly be
coupled
iii. Enzymes create
mechanisms or
environments in which
that reaction can take
place (some sort of kinase)
iv. Allows you to obtain
intermediates more easily
v. Enzymes make
intermediates more
favorable
ATP, Energy, and Thermodynamics
A. ATP Structure – Fig. 16.22
a. Sometimes ATP is written as AT~P to denote
that there is so much energy within the
phosphoanhydride bonds
b. ATP is high energy – how favorable hydrolytic E
is
c. Provides a source of energy (“energy
currency”)
d. Why are ATP Phosphoanhydride Bonds “High
Energy”?
i. Charge repulsion
1. ~3 negative charges in close
proximity that repel each other
2. Taking one away takes away one negative charge which means less
repulsion/more stability/more favorable
ii. Resonance Stabilization – Fig. 16.24
1. When the phosphoanhydride bonds
break, each compnonent has 2
resonance structure that stabilize the
break
iii. Solvation Energy
1. Packing water around the
ATP/ADP/AMP
2. Deals with free energy
iv. Entropy
1. Body likes 2 (ADP + Pi) or 3 (AMP + 2Pi) pieces better than one piece (ATP)
e. Other “High Energy Molecules” – Table 16.3
i. Phosphoenolpyruvate and 1,3
Bisphosphoglycerate can be more
favorable than ATP pathways
combined
ii. Phosphocreatine (like creatine
powder) is an intermediate that can
store energy
iii. The point is that ATP is in the middle
of the list
f. Source of Phosphoenolpyruvate Free Energy of Hydrolysis - Fig. 16.25 i. Hydrolytic reactions
is more favorable
than the hydrolysis
of ATP
ii. The reaction is
driven by the ability
to interconvert enol
to keto because keto
is more stable
(favorable)
iii. Provides very
favorable hydrolysis
iv. Can be coupled to
the synthesis of ATP
B. Use of The Free Energy – Fig. 16.27
a. ATP is in the middle – this is why it is used as
“currency”
b. ATP is relatively stable in solution which is good
because we know it will be around for reactions
c. Specific Uses for ATP
i. ATP + NDP → ADP + NTP
ii. Phosphorylation of glucose and other
intermediates as well as Amino Acid activation to
amino acyl-tRNA in Fig. 16.29
iii. Activation of a Fatty Acid
iv. Enzyme in question links MP to carboxylic acid
carbon of fatty acid and becomes hydrolyzed
v. CoA exchanges AMP
vi. In cells, ATP is always active
vii. Hydrolyzing both phosphoanhydride bonds from
ATP gives us 2x the energy which we can
calculate because getting ADP and Amp is
through enzymes that convert
phosphoanhydride bonds
viii. When we are adding up E we are using equivalence of 1 phosphoanhydride bond from ATP so we can add it up in our heads that way
d. Synthesis of ATP - Fig. 16.23
i. This coupled
reaction is
favorable because
of free E
ii. Some molecules
have enough free E
to provide for the
synthesis of ATP
e. Energy for a Person
i. Simplistic summary bc of different activities:
ii. Need amt of
ATP =~ body
weight
iii. Production of
ATP under
strenuous
activity is
much higher
bc of survival
etc.
f. Maintaining ATP Availability
i. ATP concnetrations is maintained because we need it so much in pathways, brain functions, being alive in general, phosphorylation of glugocse to Glucose 6-phosphate etc.
1. 2ADP → AMP + ATP (catalyzed by adenosine kinase)
ii. Cells in body monitor the amounts/concnetrations of ATP, ADP, and AMP iii. E charge kept at about 0.9 (measure of phosphoanhydride bonds) 1. E Charge = {[ATP] + ½ [ADP]}/{[ATP] +[ADP] + [AMP]}
iv. 0.9 means 90% in cell so we can make more if needed for when you see a spider etc. You have enough ATP for any activity that has to happen
Oxidation-Reduction and Energy
∙ Glucose and other small molecules are the source of energy
∙ As they are degraded to small molecules, energy is harvested to produce ATP ∙ Most of the reactions are not transfer reactions that produce ATP directly, but rather reactions that result in reduced electron carriers (NAD and FAD)
∙ Can come in multiple forms
∙ Usually directly provides E for ATP synthesis
∙ Reduced electron carriers contribute their energy to the synthesis of ATP
∙ The available energy in a single oxidation step is often much greater that that needed for ATP synthesis (details in Ch22)
∙ We can obtain energy values for reactions based on evaluating the electron potential difference between reduced and oxidized molecules
∙ dG = -nFdE can be rearranged to yield the NERST equation
A. Electron Carriers Employed
a. NADH is the most commonly used electron carrier in catabolic (destroying) pathways and, like ATP, is maintained at very high concentrations
b. Unlike ATP which is used in synthesis and degradation, biosynthetic pathways typically employ NADPH as the electron donor during synthesis
Glycolysis
Section Goals
∙ To develop an understanding of the glycolytic pathway and catabolic metabolism of sugars for energy
∙ To use this commonly studied pathway as a system to study how chemistry controls metabolism and how metabolic reactions are really organic chemistry reactions catalyzed by enzymes The Glycolytic Pathway – Fig. 17.1
∙ Glucose with 2 molecules of ADP and
NAD+ each to make Fructose-1,6-
bisphosphate (F-1,6-BP)
∙ Get 2 pyruvate
∙ Citric acid cycle, oxidative
phosphorylation etc. etc.
∙ On the sides there are two possible
scenarios when there isn’t oxygen of
there is limited oxygen (anaerobic)
∙ Fermentation mechanisms to
regenerate NAD+
A. An Overview of The Reactions –
Fig. 17.3
a. Think about what is most
critical for the core of the
pathway
b. Can think of as two portions
c. Start out with glucose and commit 2 ATP’s to jumpstart the pathways
d. Need ATP around or the pathway won’t even start
e. The top part is a preparatory pat of the pathway but at the bottom is where we really harvest energy
f. Each 6 carbon sugar gives us two 3-carbon pieces
g. Later we will talk about how the electrons from NADH are used to make ATP (?) h. Use glycolytic pathway to pull all of the pieces together
B. Hexokinase – P. 597
a. First part of the reaction is catalyzed by
kexokinase
b. Is part of a family of enzymes
c. “Kinase” refers to an enzyme that can add a
phosphate group to another metabolite using ATP
as a source of phosphate
d. “Hexo” refers to 6 carbons
e. Glucose is not the only substrate for some
versions of hexokinase
f. Very straightforward reaction
g. Use of magnesium, commonly found with ATP in
the body (activates it), can use other divalent
metal ions but magnesium is usually there
h. Product is phosphate groups on 6 carbon of
glucose sugar
i. Often we draw out glucose is hemiacetal form bc
typically in this form but can also be in an open
conformation
j. Thermodynamic issues – Table 17-1
i. Shown in textbook in a different location
ii. dG value based on a study done in a rat heart
muscle (naught)
iii. If you put in other variables like amounts of
ATP present in the body, becomes much
more favorable
iv. DO NOT MEMORIZE SPECIFIC Dg VALUES – just know that it is favorable v. This step is not limited to first step in glycolysis – used for several purposes like from a metabolism perspective is activates glucose but it also is phosphorylated immediately within the cell bc this trap it there – by putting a phosphate on glucose you’re trapping it in the cell – most cells take up glucose in proportion to amount in bloodstream bc passive transportation (high
conc to low conc) and this is a way to accumulate glucose in
cells so that you have the energy
vi. Ex: Jelly donut with lots of sugars, no complex
carbohydrates, makes it to bloodstream, levels rise, cell will
immediately phosphorylate the glucose. If they don’t want more, they’re not going to have as many or any transporter structure on cell surface
C. Glucokinase in liver
a. Enzyme catalyses the exact same
reaction except in the liver – sometimes
referred to as hexokinase 4
b. Reaction is different b/c glucokinase uses
only glucose, has a Km of 10mM, and
liver supplies glucose during low blood
glucose
c. Glucokinase waits for glucose conc to be
high to phosphorylate
d. Important bc don’t need to trap glucose in cells bc its in the liver which supplies glucose to the liver (liver is responsible for sending glucose to body when you haven’t just eaten a jelly donut)
D. Phosphoglucose isomerase – Fig. 17.6
a. Need to transform glucose to fructose
b. Opening ring and making it into a ketofructose when you
close the ring back up
c. Keto group comes at position 2
d. Position 6 is still phosphorylated
e. Carbon 1 is not in the ring (drawn above)
f. Carbon 2 is the ketocaron so we have the carboxyl grop and
the CH2OH
g. Conditions – dG slightly unfarovable in regular conditions
but becomes more
faorable in the actual rat
heart conditions but its
still a guestimation
h. Enzyme constantly interconverts the sugars
i. Need to be adding in glucose and taking out fructose –
relative concentrations are essentially identical
E. Discussion – what does it mean?
a. Given Keq for phosphoglucose isomerase reaction of 0.5
(approx.) and a steady state value of 0.2 G6P, what is
steady state concentration of F6P?
b. Answer is 0.1 which makes sense b/c a Keq of 0.5 means it
is only turning half of the G6P into F6P and half of the G6P
concentration of 0.2 is 0.1
F. Phosphofructokinase – P.600
a. Under standard conditions of rat heart it is more favorable
so it is quite a favorable reactions
b. Adds another phosphate group to F6P to make Fructose
1,6-bisphosphate (FBP)
c. This is the committed reaction for glycolysis! And it is crucial for pathway to be regulated
i. Tells you that every FBP is going to be used for glycolysis
G. Aldolase – Fig. 17.9
a. Reactions are
reversible
b. Standard free energy
for this is
unfavorable and in
vivo is slightly
favorable
c. Open up ring to get
reaction to occur
i. Diagram is color coded
ii. Ketocarbon is crucial to link sugar to enzyme (lycine)
d. Preserved carbon 2 as ketocarbon
e. Preserved position of phisphate as well
f. Product 1 is Glyceraldehyde-3-phosphate
(GAP) (red)
g. Product 2 is dihydroxyacetone phosphate
(DHAP)
h. Now have two unique carbon fragments – will ultimately interconvert each one H. Discussion continued
a. Keq’ = [DHAP][GAP]/[FBP] = 2x10^-4 for aldolase – is there a concentrations of FBP where the Gap concentration would be equal? Note that DHAP is about 2x[GAP] b. Answer is about .1 mM
c. What looks like and unfavorable reactions is actually favorable bc of concentrations d. Functional situation is different than the thermodynamics
I. Triosephosphate isomerase – Fig. 17.3 P. 603
a. Makes two 3-
carbon sugars
interconvert b/w
each other
b. Phosphates end
up being in the same position even though they were on opposite ends before c. Taking ketocarbon and moving around
electrons
d. GAP gets processes farther
e. Concentrations of DHAP are higher even
though can be turned into GAP
f. End up having molecules flowing through pathway with net processing of the glucose J. GAP Dehydrogenase (GADPH)– Fig. 17.13
a. Finally harvesting some energy
b. Oxidizing the molecule
(GAP)
c. Using NAD+ as an electron
acceptor (gets reduced) this
is where we get NADH
d. Not surprising that you can
oxidize aldehyde to
carboxylic acid
e. Can add phosphate onto carboxylic acid carbon without the use of ATP, can use GADPH! f. Thioester is reactive so can be replaced by incoming phosphate group
g. 1,3-BPG is a high energy molecule that can be used to synthesize ATP
h. Have transferred electrons into electron carrier NAD3H
i. With PGK (phosphoglycerate kinase) the dG is favorable but without it is unfavorable. There is fairly good reason to believe that the enzymes are somehow near each other and fed directly between enzymes – not free-flowing
j. Is a dehydrogenase
k. The dG is close to 0 which is important because a lot of reactions in the pathway are close to 0 which has an impact when we talk about the overall reactions
K. Phosphoglycerate Kinase – Fig. 17.16
a. Reaction that makes our first ATP
b. Transfers phosphate groyp to ADP
c. Hydrolysis of this
molecule (hydrolytic
release) makes it a
favorable reaction
d. Table 1 says dG is
favorable but that the
reaction by itself is
much more favorable
e. Have made first
phosphate (have made 2 technically bc one glucose makes two 3-carbon pieces f. At this point we have paid back the cost of activating glucose
g. And have made saving of making NADH
h. Name seems like is the reverse reaction but in fact names the product (in vivo can operate in both directions tho)
i. Estimated dG is close to 0 in vivo
L. Phosphoglycerate mutase – Fig. 17.18
a. Note that 2,3-bisphosphoglycerate can
be made at low levels and serves specific
other functions in RBC
b. Net reaction is 3 to 2
c. dG is close to 0 (again) in
vivo
d. Reaction is reversible – operates at essentially
equilibrium values
e. “Mutase” = moving around/changing location of
phosphate group
M. Enolase – Fig. 17.21
a. Don’t have to know electron movements, just the
substrate (2PG) and product (PEP)
b. Reaction removes water, creating a double bond
c. Operating at
equilibrium (dG
close to 0, again)
d. By the last two
reactions we
produce PEP which is a good donor of a phosphate
group to make ATP
e. “Enolase” = removing water and creating double bond
N. Pyruvate Kinase – Fig. 17.22
a. Named for the reverse reaction that
doesn’t happen favorably
i. dG in Table 17.1 shows
reverse reaction is hard
ii. Has a Magnesium
iii. Ends up giving us ATP
iv. Two 3-carbon fragments =
net yield 2 pyruvates = 2
ATPs at this step
v. Even in vivo we can estimate
that standard free energy is still favorable
Glycolysis Summary
∙ Glucose + 2NAD+ +2ADP +2Pi → 2NADH + 2 Pyruvate + 2ATP + 2H2O + 4H+ ∙ Will discuss what happens when we don’t have enough oxygen (anaerobic)
Regulation of Glycolysis
A. General Considerations in Regulation
a. Enzyme activity can be altered,
Table 17.2
b. Think about thermodynamics etc.
with each reaction and how they
are/can be in flux
c. End up with situation in which each
reaction operates at dG=0
(equilibrium) values so you can’t moderate activity at these steps
d. Can only control pathways in which the reations have a very favorable dG value in vivo: kexokinase, PFk, PK
i. Hexokinase – high levels of G6P inhibit it, step is controlled not to control glycolysis (bc not committed)
ii. PK – inhibited by ATP, activated by AMP, PEP,FBP, and low levels of ATP in muscles particularly – also not controlled for glycolysis but for the relationship between glcose synthesis vs. degradation (need to inhibit for synthesis)
iii. PFK – main place to regulate glycolytic pathway
1. ATP is a substrate for PFK, can also bind to second allosteric site on
enzyme where ATP can modulate activity of enzyme
2. Think back to discussion of energy charge ATP kept high at 0.9
concentration, in vivo this becomes as issue, well above 0.9 causes
problems, ATP bins to allosteric site and slows down activity of PFK
3. Citrate and PEP also inhibit. Citrate – don’t have very much info
4. ADP and AMP primarily are “deinhibitors” – functioning to activate PFK
by not allowing ATP to inhibit it
5. FBP is an intermediate in the pathway (product of PFK)
6. F-2,6-P – closely related ^ and is the potent activator of PFK, molecule
made by a separate system – allosteric activator – binds to alternate site
and modulates activity of enzyme (activates) so that glycolytic pathway
runs at a higher rate
7. F6P, we won’t talk about
8. NH4+ and Pi, we won’t talk about
iv. PFK
1. Committed step
2. Control at PFK occurs through cell’s energy
3. Control at PFK occurs through response to hormonal signals
4. Allosteric modulation of an enzyme changes the Km, not the dG’ of a
reaction, therefore, the reaction is no less favorable, but a higher
substrate concentration is needed to reach maximal activity
v. Cell Energy Modulation of PFK – Fig. 17.33
1. Role of energy charge is where
there is always some ATP and some
AMP, amount of ATP is dependent
on energy charge
2. We don’t see ADP at all, as ADP
conc rises, make 1 ADP and 1 ATP
B. Flux Through a Pathway
a. Flux is determined by several factors
b. Allosteric Control (or Covalent)– Fig. 17.32b
i. With an activator like ADP we get the
structure where F6P binds really well
ii. Sidechain flips depending on active vs.
inactive state
iii. Arginine has a guanidinium group that is positively charged so it maked a favorable binding site for F6P (negative b/c of phosphate group)
iv. Inactive form – arginine is flipped so have glutamic acid that has a negative charge
v. Example of how to use allosteric modulator to alter substrate affinity and therefore binding affinity – how to regulate activity
c. Hormone Control and F-2,6-P – Fig. 18.25
i. Needed to get maximal activation of PFK in any tissue
ii. Therefore, PFK-2 production is controlled differently in different tissues iii. Enzyme that makes this is the same enzyme that degrades it
iv. Enzyme activity in liver is therefore controlled by phosphorylation
v. Covalent modification is altering activity of an enzyme
vi. Funky enzyme – has 2 activities
1. That take slow conc of F6P and adds phosphate at position 2 (molecule is the major activator of PFK in pathway we were just talking about)
2. Removes phosphate from activator
vii. In case of liver enzyme – can be particular location related to hormonal control viii. Whole system is complicated because of similarity in names
ix. Remember intermediate has two phosphates at the ends – therefore the one with the
phosphate at
the 2 position
is the
intermediate
x. Remember
enzyme
pathway
being spoken about is PFK
d. Substrate Cycles
e. Genetic Control – Fig. 18.24
i. Hexokinase is very good because it’s very
efficient (ass phosphates to trap in cell)
ii. Glucokinase is slower
iii. Genetic control bc tissues can control
which enzyme they synthesize which
changes productions
C. Hormonal Control of PFK
a. When an organism needs to activate glycolysis in a tissue is can turn on the synthesis of an allosteric control molecule (F-2,6-P0 that will bind to PFK to activate it
b. Tissues respond to hormones differently
c. Heart muscle must start glycolysis during activity
d. Liver doesn’t need to start glycolysis, and in fact must turn it off if glucose is needed in the blood
e. Hormone Control and F-2,6-P – Fig. 18.25 (again)
i. F-2,6-P is
needed to get
maximal
activation og
PFK in any
tissue
ii. Therefore,
PFK-2
production is controlled differently in different tissues
iii. Turns out there are a number of different variations of the enzyme b.c there’s the liver one, the muscles one, another one in other tissues that’s not controlled by direct hormonal modulation
iv. This kind of modulation allows us to have different kinds of activities
v. Strong area of research
f. Example of a Current Article
Anaerobic Uses of Pyruvate – Fig. 17.24
∙ There are two types of systems that deal with
O2 deficiency. Both allow organisms to
continue glycolysis for ATP production without
O2
∙ They take pyruvate and let pyruvate be an
electron acceptor
A. Homolactic fermentation, the skeletal
muscle solution:
a. Pyruvate is reduced to having a hydroxyl group
b. So now have a way to get more NAD+ so we can
regenerate/continue to use the glycolytic pathway
c. Ex: Hunters know that if you run an animal to exhaustion,
you get sour meat because of the acid and lactate that build
up and cause the muscle to be sore when the anima
undergoes anaerobic activities
d. NADH concentration builds up, don’t have NAD+, can’t keep
going with glycolysis
e. Get lactate, builds up in muscle to some extent, depending on flow of blood that will move lactate
f. When lactate gets in the bloodstream, moves back to liver and in liver, where there’s usually some oxygen, gets reconverted and carries out reaction backwards, produces more glucoses and muscles can keep functioning
g. Have different forms of lactate dehydrogenase – different tissues have different version, just know lactate dehydrogenase reaction and that it’s reversible based on levels of NADH and NAD+
B. Alcoholic Fermentation, yeast’s choice – Fig. 17.25 and 17.26
a. Carry out 2 reactions
b. The first is catalyzed by pyruvate
decarboxylase, the second is
alcohol dehydrogenase
c. Yeast can survive in quite a bit of
ethanol, but its’ competitors can’t
d. We can’t undego alcoholic
fermentation, but the
microorganisms in our bodies can
e. Acetaldehyde ultimately gets
converting to fat
i. Ex: The freshman 15 – production of fat from alcohol that gets consumed – done by reversing this reaction
f. This reaction depends on a cofactor/coenzyme. Not something we have to memorize – don’t learn structure
g. Fig. 17.27 also preview aerobic pyruvate use
i. Cofactor is what we think of as the
vitamin “thiamin” and if you don’t get
enough thiamin you end up with huge
problems because you can’t go
through a lot of metabolic pathways –
yeast don’t worry about this
C. Alcohol DH Functions Just Like Lactate DH – Fig. 17.30
a. Facilitates the same
type of hydrolysis
b. “Dehydrogenase” –
typically named for the
structure that is more
reduced – carrying electrons
c. Named for reaction from lactate to pyruvate even though we’ve
been talking about them in the opposite direction
d. Both are set up so you can keep doing glycolysis even when
there isn’t enough oxygen
D. Summary of anaerobic glycolysis:
a. Muscles, when they’re stressed get glycose to 2 lactate, get net gain of 2 ATP b. In yeast, get glucose to 2 ethanol, this is slightly more favorable
c. Remember that we gain 2 ATP in either system
d. Therefore, fermentation is only 26-31%
efficient (in vivo is better)
e. Aerobic systems are somewhat better
because they’re not working with standard
conditions in terms of ATP
concentrations/levels
Other Sugars
A. The basic glycolytic pathway is to break up most sugars, not just glucose
a. Won’t be asked on a test to walk through the reactions
b. Might be asked if a particular sugar can be used in glycolytic pathway
B. Mannose – Fig. 17.37
a. Pretty easy to be put
through glycolytic pathway
because it can be easily
turned into FGP
b. “Kinda boring” bc there is
nothing different in the pathway
C. Fructose – Fig. 17.35
a. Important bc we get a lot since we usually eat simple sugars
b. Ex: fruit is not enough to be problematic, lots of sweet beverages gives you almost too much fructose
c. A lot of fructose gets taken
up in the liver which doesn’t
have hexokinase it has
glucokinase which is specific
to glucose
d. Fructokinase
e. Aldolase gives you DHAP
and GAP without one part
f. Can use ATP at this step –
not so critical to understand
details, more important part
is
i. We have a
regulatory system
deciding how much
sugar is going to get processed immediately or going to be stored
ii. There is no regulatory enzyme here
iii. All fructose in liver gets processes
iv. If we jack up levels of intermediates by not controlling access to pathway, end up trapping phosphate in cells and don’t have inorganic phosphate, can’t make ATP
v. Serious problem
g. There are metabolic diseases where the problem is worse and situations where people with too much fructose in their diets get problems with their livers
h. Just know that liver has to use alternate pathway for fructose
D. Role of fructose and fructokinase – article
a. Example of continuous series of studies on this metabolic scheme and how it can be problematic
E. Galactose – Fig. 17.63
a. Gets in our diet through milk
b. Part of lactose which is a disaccharide
c. Biggest issue people know of is lactose intolerance where they can’t
properly digest lactose sugar and digest it into the monosaccharides
d. Are also problems with galactose itself
e. There is a specific kinase that phosphorylates the 1 potision
f. Enzyme crucial for processing is an enzyme that creates an activated
version of galactose – transfers UDP which is used to put sugars in an
activated form
g. Activated galactose can be interconverted with glucose
h. Normally if we need to
make glycoproteins of in a
lactating female who is
making a lot of lactose –
we don’t have an issue
with making lactose but
enzyme 2 is sometimes
defective so there is
evidence of individuals
who are defective in that
enzyme in their diet so
they don’t get any
galactose in their diets
(genetic defect)
i. **Galactose can get converted to glucose, can go through glycolytic pathway**
Tricarboxylic Acid Cycle (TCA Cycle)
∙ Not the next section of the book, but rn we have pyruvate hanging around and we need to do something with it
Section Goals:
∙ Explore the most significant pathway in terms of oxidations of all metabolites ∙ Also, to develop an understanding of how this oxidation is regulated, to help us examine the ability of enzymes to turn biochemical into chiral molecules and to look at the use of radioisotopes in studying metabolism – something that is first introduced in initial chapter
Introduction – Aerobic Catabolism – Fig. 21.1
A. Pyruvate Dehydrogenase Cycle
a. One point is that the way it is drawn suggests that its
part of the citric acid cycle, but its’ not, it’s completely
separate
b. A lore of Acetyl-CoA comes from fatty acid degradation
and degrading amino acids
c. Figure starts with pyruvate – all of the carbons in this
cycle get eliminated as carbon dioxide
d. The second point is that they have chosen to put 2
enzyme linked intermediates on this diagram
e. If she asks to draw out cycle, don’t have to write out
these intermediates
Pyruvate Dehydrogenase Complex – Acetyl CoA production
A. Fig. 21.6
a. The enzymes in this set of
reactions are part of a huge
multienzyme complex
b. Figure is an overview of
what is happening in
pyruvate dehydrogenase
activity
c. TPP – cofactor we already
talked about
d. Release a carbon dioxide, very similar to yeast enzyme
e. 2 carbon fragment is transferred to E2 and is oxidized with electrons transferred into E2 then remaining 2 carbons get released as Acetyl-CoA
B. PDH is a Multienzyme Complex – Fig. 21.4
a. This is a huge comples set up so you can process a lot of pyruvate at the same time b. Has E2 core and E1 & E3 shell
c. E1 = Pyruvate dehydrogenase (PDH)
d. E2 = Dihyprolipoyl transacetylase
(orange)
e. E3 = Dihydrolipoyl dehydrogenase (pink)
C. Five Coenzymes are Involved
a. TPP – bound to E1
b. Lipoic acid – covalently linked to E2 - huge arm to move things from core to shell c. Coenzyme A (CoA) – substrate for E2 - activates pyruvate and allows it to bind d. FAD – bound to E3 coenzyme - is an electron carrier
e. NAD – substrate for E3 - soluble cofactor that is the ultimate place to which hthe electrons are transferred
f. ***One more thing is thing is that all of the processing of pyruvate we’re talking about happens in mitochondria***
i. One of the things that has to happen is that the pyruvate has to get in there
D. Thiamine pyrophosphate (TPP) Bound to E1
a. TP operates almost exactly as in pyruvate decarboxylase discussed in yeast fermentation except that the two carbon fragment here is transferred to the lipoamide cofactor
b. In both cases the function is to decarboxylase pyruvate E. Lipoic Acid covalently linked to lysine in E2 – Fig. 21.7 & Fig. 21.8 a. In transfer process, electrons still in 2 carbon
fragment get moved so they end up in enzyme
(lipoic acid), carbon gets oxidized
b. Rn it’s a thioester, but before it was a ketone
c. Don’t need to know structures again
d. Electrons gives you reduced form
e. Depending on version of enzyme – have 1 or
several lipoamide side chains (cofactors attached)
looks like a long arm – allows you to move 2 carbon
fragment around in the compound
F. Kidney PDC – Fig. 21.11c
a. Information is based off of bovine
kidney
b. Taken a combination of structural
studies and have put together a
model
c. In our enzyme complexes we have
an additional subunit that plays a
role in linking this together
d. Initially didn’t have E3 but now
we do
e. Large complex and having arm is
useful if you have components outside and buried
f. Ex: Another interesting thing is that lipoic acid is really crucial in terms of arsenic poisoning, inhibits lipoic acid. Darwin – took medicine accidentally – and napoleon –
probably poisoned by his wallpaper – arsenic stops
processing of glucose and the TCA cycle proper – don’t have the ability to use it
G. Coenzyme A (CoA) Substrate for E2 – Fig. 21.2
a. CoA is a way for enzymes to recognize that we have the
activated acetate
b. It’s an activation group, a way to store energy
c. Other molecules can be linked to CoA
d. Accepts the acetyl group from the lipoamide leaving
reduced lipoamide
H. Flavine-Adenine dinucleotide (FAD) Bound to E3
a. It is reduced by lipoamide so that the lipoamide
becomes reoxidizes to the disulfide structure that can
participate in another reaction
b. This is a standard FAD coenzyme electron carrier
I. Catalytic Reaction – Fig. 21.14
a. E3 and FAD are transient and (don’t
memorize electrons) extended arm can
move between active sites of enzymes of
complex and becomes associated
transiently with active site of E3, loss of
proton, rearrangement, so by this stage we
can have cofactor reoxidized and electrons
sitting in FAD
b. Get an NAD+ that accepts electrons for this
scheme (soluble e- carrier)
c. Unusual reaction because normally we
don’t move from FAD to NAD+, but in the
case of an enzyme bound electron carrier,
the enzyme active site has a huge effect on its properties – redox potential d. For a family of enzymes that carry FAD and are moving electron at redox state from keto-aldo carbon for oxidation of carboxylic acid, allows for favorable energy J. Nicotinamide adenine dinucleotide (NAD) Substrate for E3
a. NAD is, again, a classic electron carrier also used throughout metabolism b. It is mobile and removes electrons from enzyme-bound FAD, regenerating the enzyme c. This is one example where FAD donates electrons to NAD
d. Usually it is the other way around
K. Regulation of PDH – Fig. 21.17a
a. The local cell situation can
regulate PDh using both NADH
and Acetyl-CoA
b. Without oxygen, NAD hbuilds up
with no place to deposit
electrons, lipoamide cofactor
stays in a reduced state, enzyme
process slows down/stops
completely depending on how high concentration gets
c. If there’s oxygen and Acetyl-CoA isn’t getting processoed, slows down again b/c CoA levels will be low bc A-CoA levels high, hard to transfer out A groups
d. 2 products of the comples that negatively regulate activity of the complex, simply by slowing down the rate
L. Eukaryotic PDH Regulation p Fig. 21.17b
a. Mammals
b. Regulation here is still
based on cellular needs,
not on hormonal control
c. Dependent on various
activators/inhibitors
d. E1 is active in
unmodified form, if it
gets phosphorylated,
then it is inactive
e. Each E1 is separately
regulated by this system
f. Mammals have 2 extra enzymes, pyruvate dehydrogenase phosphatase and pyruvate dehydrogenase kinase
g. Kinase activated by the two products we just talked about, inhibited by low energy situations such as high concentration of ADP and moderately high levels of calcium h. Phosphatase modulated a lil bit so that its often fairly active and you have to keep having the kinase functioning if you’re going to keep E1 inactive otherwise phosphates will keep being removed by it
i. In our cells there are two mechanisms, this one and the covalent modification of E1 The Citric Acid Cycle – Fig. 21.1
A. Ex: Extra sugar from coffee, doughnut,
etc. This patheway is functioning
a. If it’s not glucose and its been
more than 12 hours since
you’ve eaten it, most likely A
CoA is coming from
breakdown of AA
b. Intermediates are regenerated
in the process – don’t have to
remember two enzyme linked
intermediates when
memorizing stuffs
B. Citrate Synthase – Fig. 21.19
a. Takes Oxaloacetate and A-CoA
b. First step is going from top to
bottom and second and third step
are on right hand side of slide
c. The way in which this works is
always from the same face of the
oxaloacetate
i. In organic chemistry, using
metal or other catalysts,
don’t normally have that
opportunity
ii. In enzymes, it’s very directional – citrate synthase is making a pro-chiral molecule
iii. Think of it as sitting down on a flat surface, never have it flipped over
iv. Bc of this, the two apparently identical conformations of central carbon can actually be differentiated both in terms of making and using in the next step d. Release of product from enzyme relies on hydrolysis of the CoA
e. Reaction is very favorable both in vivo and test tube
f. If oxaloacetate concentration is super low, need to figure out how to make reaction favorable and this is one way, by having very favorable free energy, end up combining two reactions
C. Aconitase – Fig. 21.20
a. Looking at C3 of citrate and pro S arm
with carboxylic acid and R arm
b. If the enzyme needs to associate the
carboxylic acid on C3, and the hydroxyl
groyp with the iron sulfur cluster, you
can’t actually rotate this around, it
doesn’t work – kinda complicated
i. Think of it as your hands, palm is
C3 etc. can’t flip around hands
ii. Taking molecule that isn’t chiral
and bc using enzyme, are able to
specifically recognize 2 “chemically identical” molecules
c. Moves hydroxyl group
d. Removes water and we get double bond – bond between C2 and C3
e. What happens within enzyme is once you do that it is very favorable to flip this molecule within the enzyme (look at plains)
f. Can then add water to double bond and now have carboxyl group on C2 instead of C3 g. Totally reversible, goes both ways
h. Not a large driving force either way
i. Operates close to equilibrium
j. Contonuously making citrate and using isocitrate
D. Isocitrate Dehydrogenase – Fig. 21.21
a. Moving hydroxyl group,
always moving it in this
direction
b. Always know which carbons
came from oxaloacetate
(1,2,3,4) and which came
from A-CoA
c. Uses NAD+ as electron
acceptor
d. Favorable reaction, helps
drive that other reaction
e. Gives you intermediate
f. Releasing CO2
g. If we get complete oxidation, we give 2 CO2, first from this reaction
h. Can’t oxidize carbon without rearranging carboxyl group
i. First real harvest of energy
E. Alpha-Ketoglutarate Dehydrogenae – Fig. 21.1
a. Second energy harvest, favorable reaction, the enzyme is just like the PDH complex except without the regulatory subunits
b. Bottoms 3 carbons essentially pyruvate
c. Works same way as if it were pyruvate
d. E2 is very similar to E2 from pyruvate
dehydrogenase complex (PDHC)
e. Except accepts 4 carbon fragment, releasing CO2
f. Transfers remaining carbons onto CoA
g. E3 from AKGDH is the same because you just want
to get electrons from enzyme to NADH
h. Complex is basically identical to pyruvate
dehydrogenase complex, E1 and E2 are different because substrate and there is no covalent regulation
i. Favorable reaction, so again are in situation where we’re helping to drive forward this reaction
F. Citric Acid Cycle Slide (again)
a. Both carboxylic acid carbons are from oxaloacetate
b. The fact that we have enzymes that create and recognize prochiral molecules allows us to know where the carbons came from
c. Will walk through how we figured that out, later
G. Succinyl-CoA Synthetase – Fig. 21.22
a. Named for reverse reaction, not making succinyl CoA, we’re using it
b. Enzyme puts us in a position where we can use the nergy from the thioester, transiently binding, displaces CoA with Pi
c. Pi soluble in mitochondria
d. Phosphoester hydrolysis is favorable
e. Replace one good
leaving group with
another good
leaving group
f. Second step allows
succinyl phosphate
to interact with
active site on
enzyme and active
site displaces
succinyl group
giving us a
phosphate linked
enzyme
g. Now on histidine side
chain
h. Succinate released
free
i. Ultimately enzyme
with phosphate on it
can bind a nucleotide
and GTP is equivalent
to making as ATo
j. Used tioester to make “ATP” is just counting ATPS
k. Have acquired succinate
H. Succinate Dehydrogenase – Page 811
a. This step is slightly unfavorable but not so bad that it won’t happen b. FAD operating at nomrla redox potential
c. Hangs out in mitochondria
d. Can use for energy later
e. Because of the double
bond, molecule is more
or less flat (across
carbons), understand
that we no longer can
tell the difference
between the lil dudes,
which one was from
Acetyl CoA and which
one used to be
oxaloacetate
f. Have acquired energy
g. Have mixed up the
carbons
h. Succinate to fumarate
i. Fumarate is always a trans double bond
j. The enzyme is able to specifically abstract the electrons and protons from opposite faces in a way that you always get the trans product
k. The trans aspect is one of the things you want to keep in mind for structures**** I. Fumerase – Fig. 21.24
a. Can add water across double
bond
b. Diagram is in textbook – had
good experimental evidence
that there were two separate
pathways. Nice example of
trying to figure out
mechanism
c. Outcome is malate
d. Operates ~at equilibirum with
fumerase
e. Fumerate is converted to
malate with hydroxyl group
and hydrogen
f. Very close to oxaloacetate
J. Malate Dehydrogenase – Page 813
a. (S)-Malate to Oxaloacetate
b. Looking at the oxidation of the carbon
c. Produce NADH
d. Actually what’s happening is that the NADH
is carrying 2 electrons and a proton
e. Oxaloacetate is not typically present in high
concentrations
f. The way in which the cycle is able to
continue is that citrate synthase is a very
favorable reactions because you are able to
hydrolyze off the CoA, and the fact that its
so favorable that even in the case where the previous reaction is unfavorable is driven by this reaction being so favorable
g. Also, the coupling, even though out of order, these enzymes are associated, not covalently bound, so the fact that one is right before the other makes sense. Channeling small amount of oxaloacetate right into next reaction
K. Citric Acid Cycle Summary
a. All is net energy recovery from the
oxidation reactions
b. Actually getting a lot of yield from
energy
c. Book says a few words about what
that means in terms of ATP, she’s not
going to do that at this point bc it’s a
little more complicated than
absolute #’s so we’ll talk about it
later
d. For now, just know yields of reduced electron carriers and GTP & ATP for each portion of the scheme
L. Discussion on Isotopes
a. Q: Remember the prochiral discussion? Where would the triangle and star be on a ketoglutarate?
i. Star is on CH3’s on pyruvate and ACoA and the triangles are on the carbons next to those
ii. On citrate the star is on the carbon 1 down from the triangle (Fig. 21.1)
iii. A: The triangle is on the top carbon, the star is right underneath.
b. Q: What about on oxaloacetate?
i. A: Reaction run in a pool of molecules. In fumarate, can’t tell difference
between 2 carboxylic acid carbons. Triangle is split between the two locations
(top and bottom). The star is on either of the two middle carbons.
c. If we take labeled oxaloacetate and go back into the cycle again, are we going to release the two triangle carbons are CO2? Yes. Will be released in next cycle. What happens then to those carbons as well?
d. Isotope chasing:
i. First time around w/ newly added ACoA, released as free carbon dioxide
e. Starred carbon from a-ketoglutarate is released as CO2 etc. The CO2 released in the step before that is also from oxaloacetate
f. Should help you learn the TCA cycle better too and what is actually happening Regulation – Fig. 21.25
A. Overall perspective: intracellular considerations, no significant
hormonal control
B. A-ketoglutarate dehydrogenase is essentially the same E3 as
pyruvate dehydrogenase that a-ketoglutarate dehydrogenase,
reaction at bottom left is similarly regulated
C. High concentrations of calcium activate
D. High levels of NADH negatively regulate isocitrate
dehydrogenase
E. ATP also negatively regulates isocitrate dehydrogenase
F. Look at diagram to find negative regulation
G. NADH build up ultimately turns off not just pyruvate dehydrogenase and turning on lactate dehydrogenase, but also you will get complete back up of TCA cycle
Amphibolic Nature – Fig. 21.26
A. Make or eliminate Citric acid cycle
intermediates
B. Just said excess lactate makes way to
liver and in liver is converted to glucose
C. In order to make that happen, actually
need TCA intermediate and in a week or
so will get to glucose synthesis and the
pyruvates needed to make glucose, its
ready to go from lactate to pyruvate but
its not easy to go backwards
a. Process happens b/c of 4
carbons fragments to go back to
get glucose and we get to 4
carbon fragments from
pyruvate, so temp addition of
carboxylic acid carbon
b. This particular reaction, if using
TCA intermediates to make
glucose, will require us to make
new TCA intermediates
D. There are other reasons why you would
need other TCA cycle intermediates
a. The other thing that should be obvious is that there are AA all over the place (in green) – in black means more than one can come this way
b. Basically, when we consume protein we have way more AA than we need
c. Carbons get broken down and used in fat for storage or (sssomething else). The vast majority of the AA get converted into TCA cycle intermediates on their way to fat synthesis
d. Worth recognizing the fact that a lot of input in the TCA cycle is through AA E. Can also use the TCA intermediates to reform AA
a. Last use is the use of citrate for fatty acids and cholesterol
b. Basically what is going on is that citrate is used for taking the ACoA that’s produced from AA breakdown, glucose breakdown etc., attaching it on so its part of citrate, shove into cytoplasm (in mitochondria) and in cytoplasm the two components of ACoA get reunited
c. The 4 carbon part of the citrate gets shoved back into cytoplasm and gets reused d. Citrate is essentially a carrier to get ACoA into cytoplasm where __ is produced F. Constantly in flux, a significant thing it’s doing is giving us energy from foods we consume G. Also heavily involved in some other activities
a. What happens when we have low TCA cycle intermediates and have a need to run the TCA at a more rapid rate?
H. Oxaloacetate From Pyruvate – Fig. cont
a. Anaplerotic
b. If you have a lot of ACoA, you’re either carrying out a
lot of activity but you don’t have a enough TCA
intermediates
c. Its possible that what you need to do is store some
ACoA, but need to attach it to citrate to shove it into
cytoplasm
d. So high levels of ACoA shows we don’t have enough
of the intermediates
e. So we has pyruvate carboxylase, activated by high
levels of ACoA, provides a way to make oxaloacetate
from pyruvate and ATP and CO2
f. Use of CO2 in this reaction is essentially temporary, transiently using the CO2 so that we have more intermediates but that specific carbon gets released at CO2 in TCA cycle activity so it’s considered a temporary use of CO2
g. This particular reaction is crucial to allow the TCA cycle to function at optimal levels without always having lots of intermediates that aren’t otherwise necessary
h. Turns out also that b/c of production of oxaloacetate, can use that as first step in production of glucose, can take pyruvate (liver in conditions where it has lactate), can easily be converted into pyruvate, just saw how pyruvate can be turned into
oxaloacetate, oxaloacetate can favorably be turned into malate, and then that can be turned into glucose ~~ woo provides building block that can be used for the synthesis of glucose
i. This reaction and its control turn out to be a crucial mechanism
j. Make a point here that we’ve just walked through 2 most intense cycles that will be on first test. These are the 2 BIG pathways. Start thinking about best way to have all of that in your head. ☹
Glycogen and Signaling
Section Goals:
∙ For students to develop an understanding of the
importance of carbohydrate metabolism in the
body and the mechanisms by which carbohydrate
storage is regulated
∙ Another goal is to use the hormonal control systems
involved in glycogen metabolism to illustrate
general aspects of hormone function
Glycogen Metabolism – Fig. 18.1
∙ Grey ends are non-reducing, addition and removal
of glucose happens at the non-reducing ends
∙ There is one reducing end where the whole things
starts, turns out this end is tied up in a protein so
you can’t oxidize this end
∙ Similar to structure of starch except that glycogen has lots of branch points and starch does not ∙ Each purple circle is a glucose with two chains growing off of it
∙ When you’re either adding glucose or taking them off when needed, have lots of places where you can remove glucose residues
∙ Good for bursts of energy b/c readily available for rapid activity
Why store glycogen?
∙ Speed
∙ Rapid source of energy
∙ Anaerobic metabolism Muscles that
store glycogen can carry out a
significant amount of glycolytic activity
without the availability of oxygen
∙ Blood glucose-brain function
∙ Brains need glucose to function and if
we were dependent on just the
amount of glucose in our bloodstream
we would have to eat constantly,
neurons use a huge amount of glucose so need to have glucose available
∙ A lot of the ATP made from glycolytic pathway is used to maintain ion concentrations across the membranes of the neurons (signal firing) so bodies work really hard to maintain ∙ ~Constant amount of glucose in bloodstream, easier with a store of it in the form of glycogen. ∙ Maintaining blood glucose levels happens in the liver and also sometimes some other tissues ∙ Osmotic pressure is low
∙ Each individual molecule causes a small
amount of osmotic pressure, glycogen
reduces osmotic pressure b/c its one
molecule
Glycogen Structure – Fig. 18.1
∙ All a-1,4 and at branch point a-1,6
∙ The one position is linked with either 1,4 or
1,6 which is why there are so few reducing
ends
A. Glycogen Degradation – Fig. 18.2
a. Carried out by glycogen phosphorylase
i. Does so in a way that preserves some of the energy
that is there
ii. Has a complex structure
iii. There is a piece of glycogen that binds to maintain
the association of the glycogen with the enzyme
(helps with speed)
iv. Contains active site region that has a cofactor
v. Contains allosteric regions important in modifying
system
b. Debranching Enzyme
i. Plays role in breakdown of branches to help you get to second tier end c. Phosphoglucomutase
i. Required so that phosphate ends up at 6 position of glucose, critical for beginning of glycolytic pathway
d. Glycogen phosphorylase – Fig. 18.3
i. Has an active site with a
cofactor based on vitamin B6
ii. Enzyme binds a nonreducing
end in active site and a Pi as a
substitute for the a-1,4
linkage so you have the one
position linked to a Pi
iii. Glycogen ends up 1 residue
shorter
iv. Product released is glucose-1-
phosphate – not the
intermediate for glycolytic
pathway
e. Phosphoglucomutase – Fig. 18.4
i. Moves phosphate
from 6 to 1 position
ii. Functions at
equilibrium
iii. If you’re degrading
glycogen, there is a
constant production
of G1P and if you
have a lot of G6P it
will convert it to G1P
iv. Think of as an
intermediate in the
pathway of either synthesis or degradation
v. Enzyme works by having phosphate stuck to it all the time – so a lot like mutase that shifts locations of phosphate
vi. Now have it in G6P form
f. Debranching enzyme – Fig. 18.5
i. Has 2 separate functions
ii. GPhase is a big enzyme so can’t get into tight
branches
iii. Debranching enzyme takes a chunk of “limit
branch” and attaches it onto the basic chain
that’s closest
iv. Energetically favorable
v. Can also hydrolyze off the a-1,6 linkages, does so because a-1,6 linkages are less favorable when you hydrolyze them
vi. dG for a-1,4 is more favorable so it’s not favorable to transfer a-1,6 onto end of chain so is hydrolyzed as free glucose
g. Majority of glucose residues are released as free glucose, then has to be phosphorylated, can be sent out to bloodstream directly
B. Glycogen Synthesis
a. Glycogenin (start a new glycogen)
i. People used to think glycogen phosphorylase worked backwards
ii. Disease of glycogen metabolism where glycogen phosphorylase doesn’t work yet these people have normal glycogen – basically have trouble with rapid
activity b/c can’t breakdown glycogen
iii. Led to another enzyme – glycogenin (not the one they discovered in response to disease)
b. Phosphoglucomutase Glucose-6- phosphate -> Glucose-1-phosphate
i. Need or can’t get G1P
c. UDP-glucose Pyrophorylase
d. Glycogen Synthase
i. The enzyme that was discovered in response to metabolic disease
ii. Adds glucose residues onto glycogen
e. Branching Enzyme
i. Makes branches
f. Glycogenin – Fig. 18.8
i. Catalyzes same reaction as glycogen synthase
ii. Depends on UDP glucose
g. UDP-Glucose pyrophosphorylase – Fig. 18.6
i. Have UDPGPP
ii. Have UTP as nucleotide providing energy
source
iii. Just like GTP situation from before (TCA),
this is equivalent to ATP so UT P provides
energy
iv. Linking phosphates b/w G1P and UTP and
releasing UDP-Glucose (only has 2
phosphate groups)
v. The Pi in vivo is immediately degraded
which makes the reverse reaction
irreversible/impossible
vi. Now committed! Favorable reaction
because use up phosphoanhydride bond
h. Glycogen synthase – Fig. 18.7
i. UDP-Glucose is an activated sugar – provides the energy, glycogenic or glycogen synthase, either way is transferred through enzyme into a-1,4 linkage and 2 phosphate residues end up with UDP
ii. Always couple the
addition of
phosphate onto
glucose (hexokinase
or glucokinase) with
this reaction then
get ADP and UDP or
to think about total
number of free
phosphate released
from the scheme,
can do by going
back to previous
reaction where we
released Pi
iii. Net use of ADP is 2
iv. Ended up recovering ADP or UDP
v. For each glucose added onto glycogen, have used essentially 2 ATP (1 ATP and 1 UTP)
i. Branching enzyme – Fig. 18.9
i. Makes branches
ii. Segment has to be reasonably
large but not 2 large (6 long is
averageish)
iii. More branches you have, the
more glucose can be released
very rapidly
j. Energy differences
i. Glucose to pyruvate gives 2
ATP
ii. Glycogen to pyruvate, short
term net 3 ATP, but more
energy is used in synthesizing glycogen when energy is available
iii. Short term 3 comes from breaking down giving G1P, already phoshphorylated aka don’t need hexokinase and don’t need ATP for that first step
iv. Has significant advantages b/c that’s 1.5x as much energy
v. If you’re gonna release glucose from liver it needs to be free glucose k. How can we control so many processes happening in cytoplasm?
i. Ex: If you eat a lot of breakfast before physics, some glucose is being stored as glycogen, if you haven’t eaten since last night, body is
still maintaining blood glucose level for brain so it is
degrading glycogen made last night. Controlled
through specific enzymes Glycogen phosphorylase and
synthase
C. Allosteric regulation – Fig. 18.10
a. Scheme tells us a little about the regulation, not really about the signaling b. A lot of things are put into this figure
c. Allosteric changes between active form and inactive form
i. “R form” is active form
ii. “T form” is inactive form
iii. Typically have just these 2
conformations
iv. In the case of GPhase, also
have the ability to make
covalent modifications to
enzyme that can change it
activity
v. Phosphorylase kinase
phosphorylates the enzyme
vi. There is serine in the
enzyme that gets
phosphorylated by ^ and
dephosphorylated by phosphoprotein phosphatase
vii. The modified form of an enzyme that is enzyme is usually referred to as a small “a” as in “phosphorylase a”, unmodified and less activated is “b” as in
“phosphorylase b”
viii. Glucose can bind to phosphorylase (GPhase) and puses it to inactive form even though its phosphorylated
ix. Acts as a control pathway
x. On nonmodified version of enzyme, can further push active conformation to inactive conformation if you have G6P or ATP, if you have high levels of AMP, can push inactive to active, releasing more glucose from glycogen
xi. So have enzyme whose activity can be modified by a wide variety of inputs xii. Phosphorylase kinase is very regulated
d. T vs. R Conformations – Fig. 18.11
i. N terminus of T state is
down in the back but in
R state is up and pulled
away, making it more
accessible
ii. Green loop in T state is
peptide backbone of
protein and is blocking
access to active site
iii. R state does not have
green loop in the way so
active site is much more
accessible
iv. AMP is an allosteric activator, bound to R state
v. If ATP is bound, extra phosphates can cause problems and push it back to T state
e. Signal Cascade – Fig. 18.12
i. Representative of a
number of control
systems, she has named
the enzymes we care
about in our pathway
ii. Have covalent
modifications of an
enzyme that polay a role
in activity changes
iii. Fairly straightforward
cyclic scheme
iv. Phosphorylated is more
active w/ tiny “a”
v. How much active enzyme you have depends on the kinase that phosphorylates it as well as the phosphatase that inactivates it, each has a control mechanism vi. At the bottom they’ve written out the two reactions you have to consider when thinking about whether the enzyme you’re looking at is going to be
predominantly active or inactive (depending on phosphorylation event) vii. Even more complicated b/c getting both those enzymes in their active forms is a whole other additional issue
viii. Can have multiple cyclic cascades that just adds one more level of regulation f. Bicyclic Cascade – Fig. 18.13
i. A bicyclic cascade forming another level of complication
g. Overall control of glycogen, muscle – Fig. 18.14
i. Glycogen is only half the photo and to the side is active form and there is another letter designation, “o” = original, “m” = modified
ii. On the same level of glycogen phosphorylase there is glycogen synthase
iii. GSynthase exists in
both and active
and inactive form,
regulated through
same cascade as
GPhosphorylase
****buttttt for
glycogen synthase,
it is more active
when it is not
phosphorylated**** Original form is active
iv. Think about this from a logical perspective – situation in your muscles v. EX: ran down hill and exerted muscles. Had to turn on glycogen phosphorylase Want only one to be on while the other is off.
vi. Phosphorylase kinase is in charge of phosphorylating
Glycogen phosphorylase
vii. In its original form it is less active, phosphorylation is active
viii. Multiple steps go into this enzyme being
activated/deactivated
ix. The alpha and the beta subunits can both be phosphorylated,
essentially helps remove an inhibitory segment that blocks
the active site
x. The gamma subunit of glycogen phosphorylase can be
completely removed and it will still function – delta subunit is
also an inhibitory subunit, works through sensing calcium
1. Ex: Running downhill, know muscles increased level
of calcium. Need to remove inhibitory subunits and have the other part controlled by calcium and others by phosphorylation. Maximal activity comes from when all is activated
xi. Can have partial activation by hormonal control etc.
xii. Glycogen synthase has a control mechanism that is more complicated than that of the phosphorylase mostly because if you follow yellow/orange activation lines, you’ll see only phosphorylase kinase activates glycogen phosphorylase, but a bunch of other things can activate glycogen synthase (like protein kinase A)
xiii. The bottom half of the slide has purple lines that represent dephosphorylation xiv. Protein kinase A at the top is the enzyme that is the easiest to think about in terms of being a direct link to hormones. Other names: PKA
xv. Enzyme controlled by levels of cyclic AMP in the cell
xvi. Exists in an inactive state and an active state. Phosphorylated in both conformations so is controlled by the binding of cyclic AMP which removes inhibitory subunit from catalytic subunit so you get the active enzyme and the regulatory subunits bound up with cyclic AMP
h. Overall control (cont).
i. Phosphoprotein phosphatase controls the dephosphorylation of the enzymes from before
ii. In its phosphorylated form, phosphoprotein phosphatase inhibitor controlled phosphoprotein
phosphatase
iii. There is modulation
of the phosphatase
inhibitor by
phosphorylation
iv. So, in a hormonal
situation where you
want to have
glycogen
degradation, one of
the things that’s important is the inhibitor active, making phosphatase inactive, with a net result that glycogen phosphorylase stays in its active state longer, resulting in more degradation of glycogen
v. Control scheme is not the whole story
vi. Another way to control phosphoprotein phosphatase is its association with glycogen – not included in slide
i. PKA structure – Fig. 18.16
i. Important that we see that
there is the inhibitory part by
itself that has 2 cyclic AMPs
that changes the
conformation
ii. There is a phosphorylation site
on the catalytic site of the
enzyme
iii. Cyclic AMP activates the
enzyme
j. Back to scheme from before
i. Advantage to the bicyclic thing
is that you can get amplification of a signal
ii. Each active phosphorylase kinase can active a couple molecules of glycogen phosphorylase which can then use glycogen
iii. EX: Ten molecules active one enzyme, who
activate 10 molecules themselves, now
have 100 glycogen phosphorylase
activated. A lot at once is better than a
little trickle
k. Calmodulin – Fig. 18.17 and 18.18
i. Protein that has several helices and a helix
in between the two
l. Ca2+-CaM activation of Kinases – Fig. 18.20
i. Calmodulin site drawn at the bottom
ii. Need to open up catalytic site
iii. Have a covalent regulatory domain that needs to
be moved
iv. Little green phone thing is the calmodulin, when
calcium binds to it, it associates with regulatory
domain and causes a conformational change that
removes it from the active site, allowing for
substrate to come in
v. Phone looks funky now kinda like a music note
vi. There are many kinases regulated through the
availability of calcium, both involved in regulation
of myecin activity in the case of glycogen
phosphorylase in terms of metabolism in muscles
vii. This scheme lets us control the activity of
phosphorylase kinase in part through calcium calmodulin
viii. Need both this calcium system and the phosphorylation of both alpha and beta subunits to activate the phosphorylase kinase, optimal activation of glycogen phosphorylase and inactivation of glycogen synthase to make energy
m. Hormones on muscle – Fig. 18.22
i. Diagram helps you
understand the second
aspect of its regulation
ii. Oval represents
glycogen
iii. Subunit GM
(m=muscle) and a
subunits of PP1c
associated with
glycogen
iv. Role of subunit is to put
the phosphatase where it needs to be to function, associating with glycogen so it can have activity
v. When its associated with the singly phosphorylated it is most active, with two phosphate groups it is inactive, block its association, phosphatase is released and it can now bind to soluble inhibitor which now further controls the enzyme
vi. To make the system work, none of the binding interactions and the subunit are permanent, they are on/off
vii. Mostly have association in terms of double phosphate
viii. It’s pretty easy to understand why we would want the event to happen (double phosphorylation) because we might not want it running all the time
ix. Two phosphates means enzyme is not localized in an active way and can be further stopped by its association with an inhibitor, don’t want to keep removing phosphates if you really need glycogen degradation
x. Muscles are controlling system partially by phosphorylation of enzymes from before and partially by blocking activity of this phosphatase so that the phosphate groups stay on
xi. EX: suppose you were in UC eating a parfait, level of
blood glucose high. Leads to insulin being released.
Insulin causes a single phosphorylation event on GM1
which means any available PP1 gloms onto GM and is
associated with glycogen
and can remove a phosphate and start process of turning
system around.
xii. ^Part of the reason why this works well is that in a resting
state, the most active version of the phosphatase is the
one associated with a singly phosphorylated GM subunit. Also works b/c of diagram with green and red boxes (regulation slides) where she told us the majority of the activation was from phosphorylated but small molecules help as well.
xiii. Free glucose can force T state of molecule before. By pushing glycogen phosphorylase into T form, phosphate group is much more available and can be taken off more easily by PP1
xiv. The role of the small molecules in controlling enzyme activity is so that you can regulate activity, molecule is more likely to be inactive b/c phosphate group can be taken more easily
xv. Goal is to manage sugar so we can function
n. Hormones and the liver
i. Low blood glucose effects on metabolism
ii. Low blood glucose cause pancreatic alpha cells to secrete glucagon iii. Glucagon receptors are analogous to epinephrine receptors and activate a G protein that in turn activates adenylate cyclase
iv. [cAMP] rises activating protein kinase A – endpoint of several steps here v. PKA activates phosphorylase kinase which, in turn, activates glycogen phosphorylase
vi. G1P is converted to G6P and hydrolyzed to free glucose
vii. Glucose moves through a permease in the membrane to raise blood glucose viii. Glucose is synthesized – next section.
o. Exercise and stress – Fig. 18.27b
i. Turns out liver cells have a second receptor for
epinephrine, so in the case of fight or flight there
is also a way to increase calcium levels
ii. Liver has system that moves G6P into the ER and
makes free glucose
iii. Liver is responsible for keeping free glucose in
blood
p. High glucose vs. glucose use – Fig. 18.23
i. Glucose is stored as glycogen
ii. Glucose is degraded and fatty acids are made(F-2,6-
P is made to activate glycolysis)
q. Hormone Receptor interactions – Fig. 19.1a and 19.3a
i. Pancreas makes insulin and glucagon, different
cells in charge
ii. Secreted, end up in bloodstream and different
tissues
iii. Lots of tissues respond to these hormones,
depending, way they respond or don’t is
whether they have a receptor for the particular
ligand or hormone in question
iv. At the bottom is a graph from text to remind us
that hormone binding to a receptor can be thought of the same way as a substrate bound to an enzyme
v. Response curve creates different levels of response within cell depending on affinity of cell for hormone
r. G protein coupled receptors – Fig. 19.14
i. Cartoon belies
complexity – part of a
whole large family
ii. Turns out glucagon
receptor that signals low
blood glucose and the
epinephrine fight or
flight hormone work
through an identical
mechanism that results
in the synthesis of cAMP
iii. They have 7 transmembrane segments
iv. When the right hormone is there in good quantity and binds to receptor, receptor associates with g proteins
v. Association with G protein with the receptor causes a conformational change with G protein as well, results in GDP being lost
vi. GDP inactivates the G-protein, becomes GTP
vii. ATP comes in with the orange protein and they come together to make cAMP + PPi
viii. Phosphorylation of glycogen synthase and the other one
ix. To turn off, 1. Hormone disassociates 2. Adenylate cyclases are allosteric modulators of the G protein itself. G proteins have ability to bind GTP and also to hydrolyze GTP. Association with adenylate cyclase, there are a bunch, kinda like a timer and then the GTP with become hydrolyzed and this reaction stops bc the GDP form does not associate with adenylate cyclase.
x. Need to keep sending hormone and having receptors on surface
xi. Gives an idea about what’s happening on surface
s. Signal System – Fig. 19.17
i. Have G protein that
actually looks more
accurate, a b gamma
subunits, b and gamma
subunits are not very
relevant but play lots of
roles in other situations
ii. GDP is lost and GTP binds
rather than a phosphate
being added
iii. Include changes in gene
expression which are
primarily controlled by
phosphorylation through
protein kinase
t. Other G protein systems – Fig. 19.54
i. In liver, receptor is also
available to respond to
epinephrine so you can get
optimal regulation of
glycogen phosphorylase
ii. Mechanism is fairly similar. In
response to receptor, get
hydrolysis of a head group of
a phospholipid that contains
something bisphosphate,
produces phosphate free
thing. Trigger to allow
calcium transport into cytoplasm. Don’t need to know these details but should be aware that calcium increases can be induced by several mechanisms u. High blood glucose and the liver
i. Pancreatic beta cells secrete insulin
ii. Insulin binds to its receptors on liver cells
and other cells are sensitive (variety of cells,
need to have receptor)
iii. Liver is responsive to insulin just not with
glucose transport chains
v. Receptor Tyrosine kinases – Fig. 19.25
i. Cytoplasmic domain has enzymatic
activity
ii. Insulin receptor is the one that looks like
a spaceship
iii. Insulin binding activates kinase on the
cytoplasmic side
w. Two RTK Models – Fig. 19.27
i. Have to form a dimer to send a signal
ii. 2 kinase domains come together upon ligand binding,
phosphorylate each other
iii. All of the enzymes function to add phospjate grops to
specific tyrosine groups
x. Insulin receptor – Fig. 19.67
i. pY – phosphorylated tyrosine residues
ii. Phosphotyrosines are binding spots,
depends not on it being phosphorylated but
also the specific AA around the tyrosine that
are critical for docking
iii. For insulin receptor, most critical piece is
IRS proteins
iv. IRS – insulin receptor substrate - proteins serve as a scaffold/an adaptor. When they associate with phos. receptor, they get phos themselves, pY’s
v. Don’t need to know all the things that go on in figure
vi. In muscle and in adipose/fat tissue, one outcome of insulin binding is glucose transport. One critical player is protein kinase C. Also have to have some
modification of cytoskeleton
vii. Glycogen synthesis - muscles and liver – when there slots of blood glucose, one response is synthesis of glycogen to lower blood glucose levels (like after a
donut). Glycogen synthase can be phos and turned off by lots of enzymes, of or them is GSK3beta which gets turned off by insulin binding to receptor at top
y. MAPK – Fig. 19.40
i. Don’t learn diagram
ii. Primary mechanism by which insulin can
cause changes in protein synthesis
iii. Multiple steps
iv. Also involved in cell growth
v. Occurs in cytosol
vi. Pathway that can get turned on in abnormal
responses like to a cancer or something
vii. One of several related paths
z. PKC and other enzymes- Fig. 19.60
i. Several steps involved in activation
ii. Have a calcium interaction site that’s critical
iii. Have a site that associates with lipids
Glucose Uptake – Fig. 20.11
A. Skeletal muscle and the liver
a. In response to the active insulin receptor, more
glucose permease is moved to the membrane
b. When glucose concentrations are high (jelly donut),
you get a rise in insulin, and insulin stimulates
movement of vesicles from inside of cell to
membrane of cell
c. This protein is referred to as GLUT4 and vesicle
transport to cell membrane results in GLUT4 being
on the membrane and stays there as a response of
signaling
d. GLUT4 is a permease, moves glucose in am energetically neutral way from higher concentration to lower concentration
e. Glucose gets phosphorylated and it now trapped and can’t get out
f. Way from body to uptake glucose when there is a lot of glucose in bloodstream g. In liver, also have glucose transporters, do not have GLUT4 (only insulin stimulated GLUT), have similar transporter (GLUT2, and also have GLUT1 in other cells), but it never “hides’ as vesicles, is always on cell surface
h. Liver will also take up glucose when high concentration in bloodstream, glucose goes into both tissue types, don’t want transporter moved in liver is because when blood glucose is low, liver is shoveling glucose back into bloodstream so if you moved permease from the membrane you’d have no way to shove it back out
B. How does this work? – Fig. 20.9 and 20.10
a. Glucose binding,
conformational change,
phosphorylation
b. Multipass transmembrane
protein
c. Several parts are charged so
charged residues are then
most likely involved in
providing a hydrophilic
opening so that the
glucose/or water
d. can actually associate (rest is hydrophobic)
C. Glucose transport systems vary – Fig. 20.17 and 20.7b
a. Transporter depends on energy input
b. Because do want to take in
glucose in the wrong direction
of a concentration gradient if
its from intestines – want it
taken up for body reasons
c. Energy is in the form of a
sodium gradient
d. Symport system with glucose
coming in because sodium is
coming in along its
concentration gradient, energy
comes in from making sodium gradient
e. Other ones are ways of bringing in are uniport, symport, and
antiport
f. Necessity for insulin in terms of moving GLUT4 to membrane
D. Insulin Receptor – Fig. 19.67
a. Modification of proteins involved in cytoskeletal movement –
don’t need to know details
b. Bottom line is insulin is important for GLUT4
c. Liver glucokinase concentration rises in response to PKB – glucokinase helps store excess glucose in liver glycogen
E. High glucose (cont.) – Fig. 18.22
a. Phosphatase activity is turned on and phosphates are removed from the enzymes. Note that the phosphatase in liver is actually associated with phosphorylase rather than glycogen itself
b. In liver, the targeting
subunits for PP1 are
associated with
phosphorylase itself and
most effectively with
the T form
c. There are more
molecules of
phosphorylase than
there are of phosphatase
d. To get phosphatase activity, need to have several things happening in the liver. Need to have phosphorylase in T form – small molecules (glucose) to push phosphorylase back into inactive conformation
e. When that happens, can have serine residues and PP1 hydrolyzing phosphate off of that serine which make sit modified unstable T form
f. Lots of phosphorylates around in comparison, have most of phosphorylase in T form, pushed by glucose concentrations – when that happens the enzymes is now able to take phosphates off of glycogen synthase
g. In the liver, crucial to have high glucose availability and this response system that allows you to turn on glycogen synthesis after you’ve turned off glycogen degradation F. Insulin Summary
a. Receptor tyrosine kinase format
b. Binding of IRS1 and other signaling molecules to phosphotyrosines
c. MAPK pathway activation
d. PI3K/PKB/PDK1 and PKC activation
e. GLUT4 regulation in muscle and adipose
f. Glucokinase expression in liver
g. Phosphatase activity increases
h. (If she asks you to write out a control pathway – maybe not insulin – but more the idea of what you might you if you were to talk about regulation so maybe glycogen metabolism under certain conditions, list, story, etc. A list like this would be appropriate)
i. (should you read the book? Book is useful for a lot of things but if you haven’t been reading it the parts you should read are the ones that can explain complicated ideas like in regulation. She will not ask about material in book that we haven’t gone over) G. Discussion
a. Consider a fit individual who has recently eaten
b. Would you expect glycogen to be synthesized in either muscles or liver under these conditions?
i. Guess: I think maybe the muscles because they need to build up a store
whereas the liver doesn’t have the same urgency bc its always around
ii. Answer: Both tissues would be making glycogen, how much depends on how much sugar
c. Which enzymes would be active? Liver glycogen phosphorylase?
i. Guess: Yes in muscle cells bc need to keep glucose in the cell
ii. Answer: NO. Glycogen phosphorylase degrades glycogen so it wouldn’t be active
d. Muscle hexokinase?
i. Guess: would be active to add a phosphate group to keep the glucose in the cell ii. Answer: YES. Phosphorylates hexoses or glucose in particular in this case. Need G6P so need to go back and trace to hexokinase, so it would be active. Enzyme is not committed to glycolytic pathway
e. Liver pyruvate carboxylase?
i. Answer: NO. Converts pyruvate to oxaloacetate. If you need more TCA cycle intermediates. Not active bc have lots of energy and don’t need to make glucose rn
f. Liver PP1?
i. Answer: YES. Takes phosphate off of enzyme phosphorylase. Takes phosphate off of inhibitor. Glycogen synthase. PP1 takes phosphates off of different
proteins. Will be functioning, don’t want glycogen phosphorylase active, so we take the phosphate off. Taking phosphate group off of glycogen synthase which activates it
H. Diabetes is a defect in insulin function
a. Type I, Insulin-dependent diabetes is typically juvenile
onset and due to damage to the pancreatic beta cells,
these individuals respond to insulin
i. Damage to cells that synthesize insulin. People
that need to take insulin and manage their levels.
Those individuals are responsive to insulin, so if
you’re at the proper levels, it can be fairly easy to
manage. Autoimmune disease, so it’s the immune
system that attacks the beta cells
b. Type II, Noninsulin-dependent Diabetes is typically late
onset and more prevalent in obese individuals and those
with a genetic predisposition. Elevated levels of free fatty
acids can contribute to insulin resistance which may
develop up to 20 years before NIDDM.
i. If you catch it early, you can treat it if you can do
dietary changes. Farther into the disease is harder
to do.
I. Mechanism of free fatty acid effect – Fig. 27.10
a. Phosphorylation of serine and threonine residues.
b. Tyrosine getting activated has downstream effects.
c. Can get phosphates on serine and
threonine that make IRS 1 less
susceptible to activation, less
downstream, less GLUT4 to
membrane, less glucose uptake - net
result is difficulty in regulating blood
glucose
d. Muscle uptake of glucose from
bloodstream is super crucial, liver
can’t do it all, important to reduce
blood glucose levels
e. When were less responsive to insulin
we have more glucose in blood
stream which is bad. Attaches to
proteins, causes vascular diseases, neuropathy (loss of feeling), damage to blood vessels in eyes and kidneys.
f. There are other drugs that treat noninsulin diabetes
J. Leptin (information about fat)
a. Tells body how much fat is stored (in adipocytes)
b. Has effects on brain, in most individuals if fat levels
are too high it stops having an effect, happens in brain
and in peripheral tissues where it can be burned
c. In obese individuals, does not work as well in the brain
but still work in peripheral tissues
d. Information is best identified in chapter 27 which his
books attempt to pull tissues stuff into one chapter,
good place for review at end of semester.
e. Signaling mechanism discussed in chapter 19, don’t learn details
K. Leptin uses Jak/State Pathway – Fig. 19.48
a. Receptor system that has
kinases associated with it
but the receptor itself ifs not
a kinase
b. Depends on
phosphotyrosine
recruitment which is kinda
similar to insulin
c. Proteins recruited are
transcription factors that
move to nucleus if activated
and alter transcription
L. Thyroid hormone – Fig. 19.4
a. Look at slide
b. Control metabolic activity
c. Does so through receptor – intriguing bc made by
starting w very large protein – thyroglobulin –
iodinated on tyrosine residues
d. What you get is the side chain of one tyrosine linked
to another one w three or four iodines
e. After proteolysis, end up with aromatics with iodine
on them
f. People used to use iodized salt
g. T3 and T4, depending on how many iodines are on it
h. Binds to a transcriptor associated with DNA, inside nucleus
i. Receptor is a transcription factor, group that directly modulates transcription Carbohydrate Synthesis
Section Goals
∙ Develop and understanding of the reactions involved in glucose synthesis in response to low blood glucose and the coordinate regulation of gluconeogenesis with glycolysis and glycogen metabolism ∙ Gain an appreciation etc
Gluconeogenesis – Fig. 23.1
∙ Liver using lactate as a starting point in anaerobic
conditions
∙ Subset of muscle fibers have very little in way of
mitochondria which are muscles fibers used for
short term bursts of activity. Chickens that fly
minimally, muscles are used for short bursts,
muscles function primarily through anaerobic
metabolism, few mitochondria.
∙ The more muscles fibers you have that are
specialized for fast burst activity, the more lactate
you produce
∙ Lactate from the muscles at the top of diagram
∙ Converted to pyruvate in liver
∙ In green, a large number of AA (lysine and leucine
not included) can give you carbon skeletons they
can use to make glucose. Protein turnover. Longer it
has been or few carbohydrates, a lot of what
happens is the degradation of AA
∙ When glycogen levels get low. Enough glycogen
sores for 12 hours. By time of 12 hours, definitely degrading protein. If you had some peanut butter (high protein low sugar) AA could be used for this process. Could be dietary protein or muscle stored protein
∙ Mostly the opposite of glycolytic pathway
Overview of Gluconeogenesis – Fig. 23.8
∙ Deal with three enzymes where dG is too high
∙ Called bypass reactions
A. Pyruvate Kinase Bypass – Fig. 23.2
a. Pyruvate carboxylase turns pyruvate
into oxaloacetate, adds carbon
dioxide
b. Take off CO2 when making glucose,
PEPCK, requires GTP as an energy
source, get PEP
c. Reverse pyruvate kinase step
B. Pyruvate Carboxylase – Fig. 23.4
a. Has a cofactor – biotin,
micronutrient found in diet,
but your gut also makes it a
lot
b. Biotin is the transient site
to white CO2 is added and
is where you need the ATP
c. Phosphate donated to CO2
then CO2 transferred onto
biotin
d. Biotin on long arm, lysine
residue, very flexible, can
move from one location to
another
e. Moved again to make
oxaloacetate
C. Model for pyruvate carboxylase – Fig. 23.5a and 23.5d
a. Only way enzyme
functions is if it
exists in multimeric
form, so reaction
sites are close to
each other
b. First step on one monomer and the second on a
different polypeptide that’s in the same complex so
two sites can be close enough together
D. PEP transport – Fig. 23.7
a. In mitochondria, where oxaloacetate is
b. Diagram is designed to tell you we have to transport
components, ether the PEP or the oxaloacetate which
does not have a mitochondrial carrier (don’t learn
routes now, but will come back to it later, transport is
crucial for the movement of electrons)
E. PFK and hexokinase bypass – Fig. 23.8
a. Cant reverse phoshofruktokinase and make ATP
b. Simply hydrolyze off the phosphate group, reasonably
favorably, clearly if we were just doing the two, we
would just keep burning up ATP
c. Only happening in tissue that makes glucose, enzyme doesn’t exist in muscle
F. Glycolysis vs. Gluconeogenesis
G. How Does Blood Glucose Regulate Gluconeogenesis?
a. Control of PFK/FBPase
b. Control of Pyruvate Kinase/Pyruvate Carboxylase
H. Regulation – Table 23.1
a. These are the controls that occur in the liver
I. PEPCK
a. When the liver is responding to glucagon, transcription factors (CREB in particular) are phosphorylated.
b. This binds to CRE (cAMP response element) in the control region
of the PEPCK gene resulting in more enzyme production.
c. This helps to increase glucose synthesis.
Biosynthesis of Other Carbohydrates (Start of Exam 2 Material)
A. Glyoxylate – Fig. 23.11
a. Plants can start with fat
and make carbohydrate,
we cannot do that What
they need is only 2
enzymes that we don’t
have – isocitrate lyase and
malate synthase
b. In glyoxisome, can take
two 2 carbon fragments
and TCA intermediate and
if you have TCA
intermediates, you can
make glucose
c. Can also degrade to succinate and glyoxylate
d. Isocitrate lyase carries out this reaction
e. Glyoxylate can be combined by malate synthase with ACoA to get malate which is a TCA cycle intermediate
f. *Not how seeds get enough sugar to grow
g. Mechanisms by which plants can create sugars from fat which we cannot do B. Oligosaccharides
a. Structures that are attached to proteins
b. N-linked oligosaccharides in ER and Golgi – by far the
most commons of the bunch
c. O-linked in Golgi
d. GPI-linked in ER – not talk about these at all
e. Fig. 23.16
1. Summarizes oligosaccharide synthesis in ER
2. Lots of details
3. Oligo’s are not initially added directly to the
protein
4. Started by linking sugars one by one to a
lipid that hangs out in ER membrane and has
an interesting property that it can be flipped
back and forth across ER membrane
5. Sugar transport mechanism dolichol
phosphate has to be activated (by a phosphate)
6. There are a whole host of nucleotides used in activation
7. Turns out that when the nucleotide itself is linked onto something to serve as an activator --not ATP
8. Each sugar gets activated like activation of glucose from glycogen synthesis 9. Every sugar gets activated in a similar mechanism – using a nucleotide- sugar w phosphate group on it
10. Nucleoside triphosphate group, linking, releasing inorganic phosphate
11. Look at diagram and can figure it out
12. Gets linked to very specific sequences in a protein
i. Usually polypeptides that are in synthesis
ii. Have dolichol pyrophosphate
iii. Another flippase that moves it back out
iv. Can start process over again
13. Basic mechanism used for all N-linked oligosaccharides
14. Happening in ER
f. Fig. 23.18
1. Figure summarizes relationship
with n-linked to proper protein
folding
2. Main things to point out are
that the extra glucose residues
are a mechanism that can help
to ensure that the proper
folding happens
3. Down to a single sugar,
proteins can be processed by
one of CRT or CNX –
chaperone’s help to get folding correct and can pull off
extra glucose --if folding isn’t correct, glucose can be
re-added at that point and can start folding again
4. Should have general sense that proper folding
happens in the ER
5. Once proper folding happens there is a removal of one
of the mannose residues
6. The glycoprotein now holding n-linked is transferred
by vesicles to golgi
7. In golgi get additional processing
g. N-Linked ER in Golgi – Fig. 23.20
1. Activated sugars UDP sugars primarily but not only
2. At bottom there is a CMP sugar, etc.
3. Only know that final processing
happens with N-linked sugars in Golgi
Pentose Phosphate Pathway
A. One additional way to oxidize glucose
B. 2 crucial functions
C. Three roles for Pathway
a. Synthesis of NADPH
i. Had talked about AND as accepting
electrons in glycolytic pathway or TCA
ii. NADPH is the version of the electron carrier crucial for biosynthesis, can’t make lipids, cholesterol, or steroid hormones without it
iii. Concentrations of NADPH are like that of ATP and are kept high
b. Synthesis of Ribose-5-phosphate
i. Need because of nucleotides
ii. Want to make DNA, replicate DNA etc. need ribose
c. Interconversion of odd sugars
i. We’ll see how that interconversion works
D. NADPH synthesis – Fig. 23.26
a. Oxidizes G6P, G6P dehydrogenase,
gets 6-phosphoglucono thingy
lactone
b. Controlled by concentrations of
NADP+ and NADPH
c. Oxidized glucose can be opened
more easily than just glucose
d. 6-phospho-glucono-lactonase
intentionally opens glucose
e. Second oxidation enzyme – 6-
phosphogluconate dehydrogenase
makes Ru5P
f. Each glucose that goes into
pathway in this format, get 1 CO2, 2 NADPH, and one
5 carbon sugar
g. Regulation dependent on NADPH and NADP+
h. Starting with G6P
i. Have two oxidation steps that together result in a 5
carbon sugar and release of CO2
j. Oxidative stage is stage 1
k. Look at stage 2 the interconversion of 5 carbon
sugars so if primary goal is to make R5P we can do
that through this pathway
E. Ribose-5-Phosphate and Xylulose-5-phosphate synthesis –
Fig. 23.29
a. Lots of ribose needed for nucleotide biosynthesis
b. If ratios of NADPH for Ru5P needed are balanced,
could be only thing going on in this pathway (isn’t,
but could be)
c. If the balance of NADPH and ribose 5 phosphate is satisfied by the reactions we’ve looked at, that’s the primary way in which this pathway works
d. Often the case that you need more NADPH of R5P and don’t need the balance this pathway gives you
e. To deal with issue and for other convenience purposes…
F. Fig. 23.26
a. Have whole other set of
additional reactions that allow
us to interconvert a variety of
different sugars
b. Use two different enzymes so
just to lay out where we are,
on the right hand side have
R5P and X5P below it, created
on previous slide
c. Making 5 carbon sugars allows
us to recover all of our carbons
and glycolytic intermediates
d. First reaction is catalyzed by
transketolase – takes 2
carbons from a kept sugar (X5P in this case) these are shaded in green and the carbons are green
e. 2 carbon fragment dumped onto the top of R5P giving S7P and remaining 3 carbons from X are G3P
f. Transketolase does this 2 carbon thing
g. There is an enzyme called transaldolase which is similar to aldolase in glycolytic pathway h. Moves 3 carbon fragments so top 3 carbons of S7P (shaded in blue) dumped onto 3 carbon fragment G3P now have F6P which we now what we can do with – glycolytic or back to G6P easily
i. Get 4 carbon fragment
j. Then come back and get 1 more molecule of X5P and we use transketolase – takes 2 carbons same as first but now dumped onto 4 carbon sugar not 5
k. Get a second F6P and a G3P
l. End of process get 2 F6P and a G3P having starting with 3 5 carbons sugars --one R two X m. Useful for if you don’t need much R5P but need NADPH
n. Could be true if making lots of fat for storage (talk about in 2 weeks) could be true for other tissues that have a huge need for reducing power for biosynthesis but aren’t proliferating much themselves
Electron Transport Chain and Oxidative Phosphorylation
∙ Made NADH in glycolytic pathway
∙ Made NADH and FADH2 in CAC
∙ Sources of energy
A. Where did the electrons
come from? – Fig. 22.1
B.
a. When we break
down fat and
amino acids we
get a lot of NADH
b. This is the
mechanism or
scheme
regardless of
what kind of
energy source we
have
C. Harvesting the Energy
a. We’re not going to walk through all the
math in class
b. We have a situation where if you
completely oxidize glucose and do
calculations for the energetics of the
complete oxidation of glucose and
reduction of oxygen we end up with a
whopping amount of energy
c. Have seen so far that we’ve been able
to carry out this oxidation
d. Converting chemical energy to electrical energy
e. All energy in form of NADH and a little FADH2 as far as we’ve talked about f. Conversion of electrical energy into chemiosmotic energy
i. ETC basically uses a flow of electrons to set up a proton gradient across inner mitochondrial membrane
g. Then we’ll look at the conversion of chemiosmotic energy back into chemical energy in the form of energy
i. Reduced electron carriers making ATP
D. Electron Carrier Types – Fig. 22.22
a. Have briefly seen structures before
b. Here are several different hemes that
have this basic protoporforin structure
and various side chains
c. Iron is there, which can switch between 2+
and 3+ which is where the electron gets
carried
d. Each heme group ends up being buried
within a protein
e. DON’T LEARN STRUCTURES
f. Can exist at a variety of redox potentials
E. Monitoring Carriers – Fig. 22.20
a. Monitoring absorption
b. Take a population of
cytochromes and do spectral
analysis which allows you to
see several bands
c. Levels change depending on
talking about oxidized or
reduced form
d. Using spectroscopy to get this
information
e. When looking at entire mitochondria membranes the diagrams get more complicated F. FMN – Fog. 22.17
a. Flavin Mono Nucleotide
b. Part of the FAD+ molecule
i. TCA cycle
ii. Pyruvate dehydrogenase
c. One of the interesting things about
FAD+ and FMN, Flavin molecule can
carry one or two electrons but NAD+
and NADH needs two electrons
d. Here can get a semiquinone
intermediate which is a free radical
but is possible to get
i. Turns out for electron
transport sometimes only need to move one electron
ii. Flavin is therefore useful then
iii. DON’T LEARN STRUCTURES
G. Fe-S clusters in Proteins – Fig. 22.15
a. Iron-Sulfur clusters exist within proteins
b. Majority of structure types we’ll find
c. Another version where one iron is missing
d. Get coordination sometimes to cysteine
e. Sometimes to sulfur
f. Sometimes to histidine
g. Not showing specific charge bc clusters are managed by protein in which they exist so binding site within protein for specific iron sulfur cluster
i. Single goes between +1 and +2 state
ii. But others can be at many levels
iii. Ex: 2Fe-2S can start out as +3+3 and take one electron, can go to +3+2 or could be in most oxidized state +3+2 and then be +2+2
h. Combination between protein in which they exist and the overall redox situation and the specific range of oxidation states for any particular iron atom is
i. The iron sulfur clusters share the charge over all of them – not localized, spread H. Coenzyme Q – Fig. 22.17
a. Have seen carriers free in
solution, some bound to
proteins
b. Coenzyme Q (also called
ubiquinone or abbreviated
CoQ) is a small molecule, fairly,
that is very hydrophobic so it is
soluble in membranes
c. If you look at it, the electron
carrier part of it is actually a
small aromatic ring with a
number of sidechains and what
makes it soluble is a set of
isoprene groups
i. The image shows you one with an N at the end of it
ii. Kind of chain that exists in dolichol sugar carriers
iii. This lonng hydrophobic tail is the reason why this is a very lipid soluble molecule d. Carrier very useful because it can ferry electrons between large protein complexes that aren’t very mobile
e. Can carry either one or two electrons at a time
i. While cytochromes only carry one electron
Location is Everything
A. Location – Fig. 22.2
a. Sitting on membranes
b. Most of activity is within
inner membrane of the
mitochondria
c. Inner membrane quite
impermeable while outer is
very permeable
(impermeable to large
proteins but small ones can even get through sometimes)
d. Large shape range and small size range
B. Mitochondrial structure – Fig. 22.4
a. Rat liver mitochondrion
b. Inner membrane is in a yellow
color
c. Outer membrane in a red color
d. Cristae (folds) are in green
i. While they are continuous with inner
mitochondrial membrane, have isolated
regions
ii. Would have to have a fair amount of
diffusion in an awkward setting to get
significant material between the inner
and outer mitochondrial membranes
and all the way to this contiguous but somewhat isolated space within the cristae
e. ETC sets up proton gradient – might be somewhat different in regular mitochondria vs. rat liver mitochondria tho
C. Getting NADH There the Easy Way – Fig. 22.8
a. Critical to get the NADH that’s produced in the cytoplasm into the mitochondria b. NADH produced in aerobic glycolysis has to get in** important
c. In TCA cycle it’s already in
d. 2 different main shuttles
e. Easy way (tissue specific) - know DHAP
from glycolysis and 3PG we know as
the backbone for lipids and triacyl
glycerols (TAGs)
i. One way we can process NADH
is starting with 3PG DH
ii. Electrons get used to reduce
DHAP to 3PG
iii. 3PG shows up at inner
membrane surface
iv. (Outer membrane has pores in
it – basically anything that isn’t
a protein can come into the
mitochondria, ballpark 10K)
v. 3PG shows up at inner membrane surface
vi. Enzyme has FAD+ bound, collects electrons from 3PG, now have FADH2 vii. Carbons get re-oxidized to DHAP so it can be a shuttle
viii. Chain functions in inner mitochondrial membrane
ix. Exists in bees, also works in our brains and some other
tissues as well
x. Simple, effective, takes advantage of glycolytic
intermediates (DHAP)
xi. Electrons in FADH2 are at a lower redox potential than in NADH so there’s not as much energy there, however, used very effectively in specific locations
D. NADH Conservation and Movement – Fig. 22.27
a. This is the second, more complicated way
b. Happens in skeletal muscle and in a number of other tissues
c. Mechanism depends on having amino acid skeletons (carbon skeletons) around d. Takes advantage of several transporters in inner mitochondrial membrane e. Oxaloacetate cannot get through inner mitochondrial membrane, need malate f. Situation allows us to transfer electrons not in NADH but in malate
g. Malate DH in TCA cycle, malate is more favorable than oxidized oxaloacetate – more energetically favorable
h. Malate made in cytoplasm
i. Have a transporter which is an antiporter, transports malate for alpha ketoglutarate (AKG – at the bottom of the circle) (TCA intermediate)
j. Once in mitochondria, able to recover malate to oxaloacetate and CAC can drive this – happens by citrate synthase being so favorable
k. NADH in mitochondrial matrix, now have oxaloacetate which can’t get out – so the rest of the scheme is try to get it back out now
l. Since it would be worthless to go back to malate, now convert the carbons to aspartic acid – amino transferases interconvert alpha keto acids and alpha amino acids so we get aspartic acids because we use an amino group from glutamate and the glutamate becomes AKG
m. Aspartic acid goes through second transporter (AA transporter) which exchanges glutamic and aspartic acid across inner mitochondrial membrane – neutral exchange n. End up with aspartic acid in cytoplasm and glutamic acid we needed in matrix o. Equivalent aminotransferase in the cytoplasm so can regain oxaloacetate and our glutamate
p. The 5 carbon molecule (AKG) is just there for the ride or provide the amino group or the anti-port molecule
q. 4 carbon fragments (oxaloacetate, aspartic acid, malate) are what end up carrying the electrons
r. Have both molecules cycling together with a net movement of NADH across the membrane
s. Also move a proton because you start with both of them on one side and end with both on the other side
t. So we are using proton gradient as part of the energy to drive this
u. Gets us NADH and not FADH2 across the membranes
v. Tissues that use it can save energy bc they have NADH, stronger reducing potential molecule
w. LEARN WHICHEVER ONE MAKES MORE SENSE TO YOU -- DON’T LEARN BOTH The Electron Transport Chain – The Order of the Carriers
A. Order – Fig. 22.9
a. Can start talking about ETC
itself and this diagram does
a really nice job of lining up
various players that are
involved in the ETC and
gives a sense of the redox
differences
b. When trying to figure out if
electron movement is
favorable, take value for
electron acceptor and
subtract from that the
value of the donor and if its
positive its favorable (-dG)
c. Ex: NADH is the most
negative one we’re looking
at -0.315
i. Oxygen, ultimate
acceptor, +.815
ii. Subtract -.3 from .8 get a much more positive values
d. Instead of having a direct transfer of electrons from NADH to oxygen, have intermediates that are set up in a way so that you can use that significant amount of energy to make more than one ATP
e. There are three steps shown there redox potential is large enough where you could synthesize an ATP – not actually true but is suggested on figure
f. Each complex gives a specific redox value for the reaction that is the electron transport across the complex – redox values for specific players in complexes
g. Giant multiprotein structures
h. Succinate DH with FADH2 or transport system we just talked about with FADH2 show up at same level of CoQ so show up at lower redox potential than NADH which is why you get lower energy yield
i. Cytochrome c is a small protein intermediate that contains one heme c group i. Intermediate between complex III and IV
j. Biggest jump is at bottom and where we get biggest transfer of electrons onto oxygen to give us water
k. Have tools that are able to block electron transport at specific complexes and we will use those as how they help us understand how the ETC works
i. Each functions by blocking a particular complex function
ii. Blocks ATP synthesis from that transfer but bocks electron transfer at that
function
2/24/2017
The Electron Transport Chain – the Order of Carriers (cont.)
A. The Order (cont.)
a. Makes the assumption that the electrons are floating from carrier to carrier to carrier b. This kind of alignment/flow is what is actually happening
c. We’ll then talk about the complexes and how the e flow is moving protons across the inner mitochondrial membrane
B. Carrier Order by O2 Use – Fig. 22.11
a. Similar alignment to the
previous figure
b. Representation of the
kind of experiment one
can do to study electron
transfer in the
mitochondria (in a test
tube)
i. Specialized test
tube that has an
oxygen sensing
electrode in it
ii. Using a
particular DH as
a source of NADH as long as there is beta-hydroxybutyrate
c. Have mitochondria, supplying NADH, getting a decrease in the concentration of oxygen d. If they add some specific inhibitors, find that they can block that electron transfer e. Inhibitors function in a way that they can bypass the inhibitors if they add succinate - which we’ve talked about in TCA cycle
f. The succinate DH enzyme reaction results in the transfer of electrons to FADH2 i. Not subjected to same inhibition
ii. Can deplete amount of oxygen present
g. If we add an antibiotic that inhibits the function of the succinate DH and FAD+ system, we stop the use of oxygen and electron transfer
h. Electron source is ascorbate/vitamin C and a small hydrophobic carrier that will interact and transfer electrons to the electron transport scheme – will get additional use of oxygen
i. If we add cyanide, all electron transfer terminates
j. So oxygen is the sink and we look at different ways to mess with it
C. What is a P/O Ratio? – Fig. 22.13
a. What this means in terms of coupling this to ATP synthesis
b. Graph in textbook has been updated but we use this one for the historical sense of how this work was done initially where the expectation was that you could just relate oxygen use to ATP production
c. P/O is essentially how much ATP is made from ADP per amount of oxygen used d. The assumptions were that you would have to have whole numbers – couldn’t have partial relationship between oxygen and ATP use
e. Same sense you’d get from first figure we started out with when they had ATP directly linked to the complexes
f. Trying to measure the oxygen use which we just talked about and the amount of ATP production – running to the end of ADP in each case so you’re not making any more ATP g. In this experiment once you run out of ADP you stop the oxygen use
h. This tells you there is a significant coupling
i. If you look at the current, updated graph
i. When these
experiments are done
more carefully and get
a better understanding,
P/O ratios are not
whole integers, closer
to 2.5 and 1.5
ii. Tells you the coupling
between these electron
transfer schemes is
different than direct
interaction than a
system that makes ATP
and the electron transfer couple
iii. If you’re starting with NADH at 2.5 and succinate at 1.5 (succinate is not the only place you could get electrons at the level of FADH2 – on Weds we talked about the G3PDH system that was one mechanism by which you could transfer
electrons into the mitochondria and there are other redox enzymes)
j. Looking at last step, there seems to be an integer value of approximately 1
D. Table 22.1 Redox Potentials
a. Not just the levels at which you’d donate
electrons
b. Textbook has many of the individual electron
transfer players within each complex and she
chose not to put them on there, more of a
sense of what we were just talking about
c. Between succinate and CoQ (intermediate
electron carrier) there is very little difference
which is why you don’t get enough energy out
of this electron transfer to contribute to ATP
synthesis
d. Includes actual table too – Table 22.1
i. Each iron sulfur complexes listed is a part of
complex I
ii. Each heme b is part of complex III
iii. Basically separates them
iv. Have hemes and coppers which play a role in
electron transfer
v. As you go down list, numbers go from
negative to positive
vi. NADH is better electron donor
vii. O2 is best electron acceptor
viii. If you look at second level “complex II” there
isn’t a whole lot of difference so can’t really
contribute energy for ATP synthesis through
complex II which she also explained in the
simplified table above
ix. Also means you can’t transfer protons here bc
the redox potential isn’t large enough
E. Electron Flow – Fig. 22.14
a. This is useful in terms of a big picture of what’s going on, not in terms of what’s actually happening in the complexes
b. Each complex that has a large difference in redox potential has the ability to move protons across the mitochondrial membrane and the proton differential is what is used to drive ATP synthesis
c. Complex I spans the electron potential difference between NADH and CoQ d. Complex III spans the electron potential difference between CoQ and Cyto-C/oxygen e. Figure lays out some of the players, doesn’t tell you how electron or proton flow is being accomplished
F. Complex I – Fig. 22.14, 22.18
a. This is misleading slide
b. Structural analysis of prokaryotic one
c. Has a huge transmembrane arm and
a peripheral arm and they lay out the
locations of these
d. Because this comes from a bacterial
system it’s actually sitting in the
cytoplasm and that’s what they’re
showing you
e. If we were looking at the
mitochondrial complex I we’d be
looking at the matrix – facing matrix
where NADH is
f. Name of the complex is actually NADH CoQ reductase
i. Using NADH to transfer electrons and reduce
CoQ (give it electrons and protons)
g. Has, in general, different functionalities between arm in matrix and arm in inner membrane
h. Transmembrane arm has the easiest color scheme to figure out
i. Transmembrane helices that are purple, blue cluster, green cluster, etc. each cluster represents a protein
ii. Combined together to make the arm
iii. Have a structural relationship to proteins used to serve as antiporters –
exchange protons with sodium ions (not these, the ones that they’re similar too) iv. The idea that became popular when this first structure of a bacterial complex I was solved was that the side chain parallel to the membrane was maybe a
mechanical arm that somehow altered the conformation of the transmembrane segments that helped proton flow – not actually the case
i. What actually happens is that the electron transfer occurs in bunchy region and force is communicated to transmembrane arm
i. Protons and electrons flow from the binding
site of the NADH (at top in peripheral arm –
bunchy region) to an FMN
ii. Electrons can then flow to a series of sulfur
complexes within the various protein
iii. The protons cannot bind to Fe-S complexes
iv. Protons that come in are separated from the
electrons and essentially get dumped out
j. CoQ has a binding site at the bottom rightish and
recovers protons from a different source – not
necessarily the dumped ones - and gets electrons
from Fe-S clusters
G. A Giant molecular Proton Pump – Structure and Mechanism of Respiratory Complex I a. Pic not included in slides
b. Shows you peripheral arm on the side
c. Still based on bacterial system
d. Bacterial systems that have been characterized – are around 14-15, in humans there are 45
e. All extra subunits are involved in aspects of function including assembly and protection from environment
f. Critical because there are a couple of diseases associated with complex I– some fatal g. Cartoon suggests a slightly different functionality of membrane arm but piece most helpful to think about is that the transmembrane parts (most) have discontinuities in the middle of the membrane
h. Now seems more likely that there is a link somewhat electronic but also have mechanical changes between electron flow and the transmembrane segments of these proteins so have more work to do to figure it out
i. Diagram suggest proton flow is happening through these transmembrane proteins so the proton flow is being regulated by the electron transport but not by pulling this parallel arm back and forth
j. Net result of this is transferring 4 protons across the scheme – one for each of the locations – for each pair of electrons transferred from NADH
k. Basically for the whole movement of the electrons through this scheme, get flow of 4 protons across inner mitochondrial membrane, such that the protons accumulate on the outside of the inner mitochondrial membrane
l. Can remember direction of proton flow is that the yellow jellybean diagram does it right H. Complex I Electron Movement – Fig. 22.19a/b
a. Expansive look at the
peripheral arm
b. Shows you the series of
iron sulfur clusters
c. Can kinda see the FMN
location at the top -
where the NADH binds
d. The electrons transfer
(and FMN can transfer
one or two electrons at a
time so get 2 electrons)
e. Then one transfers down,
then the second transfers
f. Proton gained in Q is probably also occurring one at a time since you add electrons g. ^All happening in membrane
I. Complex II – Fig. 22.22
a. Several things you can see –
bound FAD+ in complex itself
b. Is succinate DH, the other
enzymes that carry out similar
FAD+ are similarly placed in inner
mitochondrial membrane
c. In each case, while they have
some transmembrane subunits,
there is no mechanism to move
protons so all that’s happening in
complex 2 is that you are
funneling electrons into the
electron transfer scheme to get
to CoQ – not collection of energy
d. dE value is very small – not enough to collect energy or
make a proton gradient
e. So, enzyme essentially just gets the FADH2 electrons and
moves them through a series of Fe-S clusters
f. There’s a coordinating heme group then they get moved
to CoQ
g. Now have this whole pile of electrons (most from NADH,
some from succinate, some from the G3P DH, oxidation
of fatty acids)
h. At this point we just end up with a collection of CoQ
without adding to the proton gradient
J. Redox Loops – Fig. 22.30
a. This is how complex III functions, it is not a pump like complex I
b. Have electrons in Q – critical in
getting electrons transferred to
cytochrome C and also getting
proton flow
c. When we talked about complex I we
talked about a pump mechanism
d. Complex III works in a redox loop/Q
cycle which depends on having 2
types of electron carriers – one
carries electrons and protons and
one carries electrons – kinda saw it
in complex I but it didn’t function
this way
e. Complex III – have a carrier that
brings in both electrons an protons
f. Delivers the electrons to an electron carrier (or maybe more than on) and dumps protons on opposite side so other than a pumping mechanism, it’s a question of where the protons come from and get dropped off
g. Picks them up on one side and dumps them on the other side
h. Electrons go to oxygen but it’s not the immediate receptor
i. Initially how people thought
complex I worked too but it’s
actually a pump
K. Complex III – Fig. 22.23
a. Dimer structure
b. Color scheme shows lots of proteins
that come together
c. Have buried all the players in this
structure
d. Can see two heme groups – one closer to inner membrane space side and one to matrix side
e. Can see an iron sulfur cluster
f. Can see heme C1 – almost in inner membrane space
g. Can see docking site for cytochrome C – only one of them docked at a time h. Some protein that are on inner membrane space, some mostly transmembrane i. A lot of the proteins extend at least one (lilac) transmembrane segment through to other side in matrix
j. The other thing they have in this diagram is an inhibitor – stigmatellin that helps us figure out how scheme works
k. On the right are the redox potentials isolated
i. Cyto bH carries a single heme b
ii. bL carries another
iii. Rieske carries an Iron sulfur
iv. If you pull the players out they don’t give you
100% in order redox values
v. CoQ for whole thing is pretty close to the Cyto
b’s, cyto c is slightly more positive that the C1
vi. Rieske is more positive than you’d expect
vii. In vivo, redox potential is not exactly the .28
shown here for isolated form
L. How Complex III works – Fig. 22.31
a. This is the first half – the top half – of the
redox loop
b. Shows you a Coenzyme 2 from complex I
or II
c. Reduced electron carriers soluble in
membrane move to binding site close to
inner membrane space, binds to where
stigmatellin inhibitor was before
d. CoQ encounters situation where there
are options for it to transfer various
components
e. No place to put 2 at a time, only one electron carrier here
f. The closest electron acceptor is the Fe-S protein and the transfer
of one electron there is quite favorable
g. Q has an interesting property – if you transfer one at a time the
redox potentials are different
h. From QH2 to QH dot (radical) is much more positive so really only
effective way to get rid of an electron is easily transferring electron
to Rieske Fe-S protein
i. Protons get dumped into inner membrane space (out of
mitochondria)
j. Rieske has conformational shift that moves the Fe-S protein away from the binding site k. Left with the radical Q with a negative charge and it’s in the vicinity so it can transfer an electron to cytochrome and the second electron transfer potential is .03 which is similar to cyto b’s
l. All close enough that you can transfer the electron
i. Shown going down as if there’s a big change but its pretty much similar
m. There’s a second Q binding site for first round
i. Second Q binding site contains a completely oxidized Q
n. Now were in the matrix side (orange) – picks up an electron so Q’s are equivalent just in different locations, associated with the protein, not just free in solution
o. Electron in Rieske moves to cytochrome C 1 and differential is not favorable i. Now have electron in C1 that’s at the inner membrane space side of the inner mitochondrial membrane and at the docking location that cytochrome c comes to bind (mobile, water soluble)
p. Cytochrome C docks very quickly and leaves very quickly – relative affinity, gives orientation and stuff
q. Electron transfers favorably from C1 into cytochrome c and Cyto C diffuses away r. Left at end of first cycle with radical CoQ, way this get resolved is cycle 2 M. Complex III (Cont.)
a. Another reduced CoQ comes in exactly
how the first did, gets to same reaction
site, transfer electron to Fe-S protein,
once it gets rid of electron moves back to
original location
b. Whole process is identical to the first one^
c. This transfer is the same as well except at
second Q binding site, see waiting for it is
the free radical Q which is a better
electron acceptor than oxidized Q
d. Picks up second electron (more favorable)
and picks up protons from the matrix
e. What we’ve done is created a scenario
where two Q’s have moved their protons
across the inner membrane space – one
has ended up in its oxidized form (namely from cycle 2) the one
from cycle 1 is completely reduced again, picking up 2 protons
from matrix and one electron from first cycle and one from
second cycle
f. QH2 is free to come in and start all over again
g. From 2 cycles have net transfer of 2 e and 4 protons into the
inner membrane space
h. There’s a little bit of motion (like in Rieske) but mostly
functioning by redox loop in complex III rather than pump system
found in complex I
N. Cytochrome b orientation – Fig. 22.23 a. What’s happening at inner
membrane space is where we
have cyto c picking up electrons
to move one at a time
b. Can also see the binding site for
Q that first comes in and the
second Q binding site which is
at the bottom of the protein
(matrix side)
O. Cytochrome C – Fig. 8.42
a. Mobile electron carrier
b. Met it in Biochem I as a heme
carrying protein that has been
used as an evolutionary clock
c. Have looked at its sequence bc its
easily available and can study to
figure out evolutionary
relationships based on sequencing
(more than 30 years!)
d. Freely soluble in inner membrane
space
e. Able to move from Complex III and
complex IV
P. Complex IV – Fig. 22.24
a. Can see in diagram a nice layout of it in relation to membrane
b. Also a dimer
c. Has a docking site for cytochrome c in inner membrane space
d. Doesn’t have as much matrix side
structure as some of the other complexes e. Buried in each side are components involved in electron transfer – can see a copper center by the aqua balls on the
top left
Q. Complex IV Electron Carriers – Fig. 22.25 a. Can see the copper and the specific
protein side chains to which its
associated
b. Have a redox potential for copper a
c. Can see heme groups at the bottom and another copper part of the complex
The ETC
A. Cytochrome C Oxidase – Fig. 22.24
B. Complex IV Electron Carriers – Fig. 22.25
a. Each carrier within complex 4
can carry only electrons, no
protons
b. Proton movement has to be
through a pump mechanism
c. The copper complex is closest to
where cytochrome c docks
d. Get an electron, shared between
two coppers
e. Get copper 1 copper 1 but without the electron you just have a share 1 and 2 f. Electrons move one at a time from
center
g. Move through a heme A and get
shuttled into complex here where they
get transferred to oxygen
h. Can’t transfer electrons one at a time
trying to get them into oxygen it just
doesn’t work
i. It has been studied and oxygen gets
associated and gets reduced one
electron at a time so there is a hypothetical pathway trying to tell you what’s going on j. Start with an oxidized center
C. Electron Transfer Steps – fig. 22.27
a. Rapidly followed by second electron and proton
b. Release molecule of water
c. Oxygen was
associated/coordinated with
the iron of heme A3 and it
came from the previous round
of the cycle
d. Have water this water release,
iron 1 copper 2
e. Now can have new oxygen
bound in coordinated with the
heme and evidence suggests a
fairly rapid reorganization of
the structure of this and allows
you to get an electron from the
tyrosine, see where the proton moved from the oxygen that entered
f. Last electron needed to get structure coordinated correctly comes from the tyrosine
g. Add electron and release water
h. Have original atom of oxygen coordinated with water
i. Donated electrons so very oxidized
j. Copper now in original format
k. Can add electron and proton and get back to starting intermediate
l. Very quick cycle – pretty hard to catch all of this in process so some of it is best guess and some of it is supported by spectroscopic measurements
m. Some protons are added to the system to become part of water
D. Complex IV Also Moves Protons – Fig. 22.23
a. Doesn’t tell us about how this causes
a proton gradient
b. Before evidence, people guessed it
moved protons with a pump
c. Have better evidence now for
complex iv but there is more
clarification
E. Bacteriorhodopsin – model – Fig. 22.34
a. Don’t learn details
b. Essentially depends on a gating
system so protons can’t blow backwards across membrane
c. Partially due to the fact that the center of the
pathway is gated in a resting state without light
d. Light activates retinal which is converted into a cis
form – basically excited
e. Now have a rearrangement of the structure such
that the proton that was up there can actually
move to become associated with the aspartic acid
f. Structural change and now a chance for second
proton to move to a pair of electrons on the
nitrogen
g. Finally able to release the proton that had moved
to the carboxylic acid carbon and have another
proton come in and take the place of the one
from the carboxylic acid and start over
h. Basic chemistry tells us we don’t just have free
protons – are actually thinking of H3O+ (not H+ even though we write it this way – bc we lazy)
F. Complex IV Channels – Fig. 22.35
a. Looks as if there are two separate channels in complex iv
b. One channel (K) we don’t have a lot of players but we probably have a water – have a situation where we can imagine with the right water possibilities a way in which we can
get protons up to the copper b or the site where the
oxygen is going to be present – no way we can align
the channel with an exit on the outside so hypothesis
is that this channel provides the protons for the
water production
c. D channel – see 3 separate potential parts – trying to
show you pathways that make sense based on what
we know about structure – have potential
connection all the way across the membrane so the
evidence at this point is that through conformational
changes that as the oxygen is being reduced ( e
coming in) have changes that allows protons/push
protons from binding site to binding site – end up
with a situation where you can imagine a series of
hops the protein can take from appropriate electron
source to e source up to exit channel and where hypothetical exit channel is we have an aspartic acid there for possibilities of a last step before it leaves
d. Another unclear thing is that in addition to asp 91 there are other potential side chains to recruit protons
e. Best evidence is that as we move electrons from center – reducing oxygen – we make some conformational changes we basically push protons through channel
f. 4 protons across membrane and were using protons to become part of the water – 4 for the oxygen
g. Decrease of protons in the matrix as were moving the electrons from cytochrome c to water as a final source
G. Super-complex I1III2IV1 – Fig. 22.28
a. Shows an additional picture in class??
b. Q binding site for complex 3 and complex 1, you can imagine
a very simple transfer instead of a huge jumping
H. The Chemiosmotic Hypothesis – Fig. 22.29
a. We’ve talked about how we move protons
b. Can calculate what that means in terms of energy byu looking at pH difference between the two locations can get a measurement of the membrane potential
c. Talking about a single charge
d. Can calculate that for each proton, costs us ~21kJ/mol to move protons out e. We’re expending a lot of energy from ETC to make the process work and to push the protons out
f. High proton concentration is a source of energy that we can use for other purposes Oxidative Phosphorylation – Fig. 22.36
∙ Can see matrix in here and what look like lollipops and they are a portion of the enzyme that synthesizes ATP – the F1 particles
A. F1 Particles – Fig. 22.36
a. Are available through sonication – get vesicles where lollipops are sticking out b. Can dissociate with urea which doesn’t
cleave bonds but changes interaction so
F1 particles get dissociated and can be
reconstituted so there’s no permanent
change
B. ATP Synthase F1 structure – Fig. 22.38
a. Has a couple of proteins part of a stalk
structure running through middle of
globular structure
b. If you look top-down at the lollipop you
can see alternating beta residues and
alpha residues in the lilac color – 3 of
those – and the way this is shown, it’s
shown also with nucleotides and each
biding site – can see 5 – the catalytic part of this enzyme is in the beta subunits c. All three betas are the same and all three alphas are the same but sometimes the structures change depending on where we are in the cycle
d. If you isolate the particle san give ATP, this hydrolyzes it
e. Tells you that the only way this is synthesizing ATP is if its associated with a part in the membrane
f. Works in an energetically favorable way
– hydrolyzing ATP
g. Isolating hydrolytic part hydrolyzes ATP
h. Catalytic part is within F1 structure
C. Top Down View – Fig. 22.39
a. Tunnel/channel part
b. Uses sodium ions so purple circles are
sodium ions
c. Shows a series that span the membrane
d. Each has a spot to bind sodium
ions/partially charged small molecules
D. ATP synthase – Fig. 22.41
a. Have cytoplasm and paraplasm – bacteria don’t have
mitochondria
b. Has a peripheral arm (orange) – made up of both a
protein that interacts with the c subunits and a long
arm that spans from the membrane up over the top
of the catalytic portion of the structure
c. Book goes back and forth between terminology for
mitochondria system and bacterial system so only
remember the parts that are the same – STALK and
PERIPHERAL ARM and that’s good
E. Diagram of Action – Fig. 22.45, 22.46
a. Aspartic acid which is a potential binding site for a
proton
b. What is believed to happen is the ring
of c subunits 10-15 and the side
protein associated in the membrane
has a way in which the proton can get
through from the matrix to the
aspartic acid in one particular c
subunit – which causes a ink, kink
pushes rotator and moves a proton
that’s gone all the way around to an
exit site in same protein from which it
can exit out of the complex to the
inner membrane space
c. So every time a proton come sin it pushes the rotator – with a lot of protons ca move rotator pretty quickly
d. Allowing protons from high concentration to associate with c proteins, kink, turn, turns stalk, stalk doesn’t turn F1 portion, rubs against it
e. A and peripheral arm don’t move, stalk has to turn against this full structure held in place by peripheral arm
f. Have looked at AA inside alpha and beta and it’s clear that there’s nothing holding them together – lubricated
g. Causes conformational changes in alpha and beta subunits which causes ATP synthesis h. Gradient of protons that is creating this rotation, causes conformational changes causing synthesis
F. Isolated ATP synthase can be turned on by a proton gradient – Self fig
a. Can mimic and create vesicles
with ATP synthase and if you set
up a proton gradient so that it is
able to let protons enter c
sections and come through
synthase, actually see ATP
synthesis
b. Can mimic without complex 1 3 4
c. Shows it’s the movement of
protons driving ATP synthesis
d. Without gradient, there is no synthesis (right side of image)
G. dpH is Critical! – Fig. 22.46
a. For liver mitochondria with a pH
difference of 0.75, DG = -21.5 kJ/mol
to move the H+ in. About 3 H+ /ATP
appear to be needed.
b. Trend negative bc favorable for
protons to come in
c. 3 protons to synthesize an ATP
d. Can take a solution of mitochondria
and add in a small hydrophobic
proton carrier and will dissipate
proton gradient
e. Get no ATP synthesis
f. People have shown it is not the membrane potential difference, it’s the pH difference H. ATP Synthase sites – Fig. 22.42
a. Alpha and beta subunits are the critical ones
b. Pointer is the stalk
c. Have three sites – open site, no binding
d. Loose site – can have initial binding of substrate
e. T site – only ATP can be here,
conversion of loose site to T
site is the catalytic force that
drives the synthesis
f. Have energy in the form of
rotation – pointer changes
conformation, convert all
sites tight becomes open,
loose become tight, open
becomes loose and allows
binding
g. Open site releases ATP that was synthesized (and water) now have additional energy we can move back
h. Happens rapidly
i. Can cause conformational changes within the beta subunits that creates this continuous cycle of synthesizing ATP as long as there is proton flow through motor
I. Three Sites are Coupled – Fig. 22.42
a. Conformation of the active sites
in the ATP synthase is modulated
by proton flow through the F0
portion of the ATP synthase and
how that drives the
conformational changes and the
changes drive the synthesis of
ATP and if we don’t have it we
just have it free in solution
b. Enzymes stabilize the highest
energy state to which the
intermediates have to get in the
reaction and so clearly its forced in a direction here that we get ATP synthesis under conditions where the proton gradient exists
J. An uncoupling proton channels – Fig. 22.47
a. In mitochondria present
in a specific type of fat or
adipose cells namely
brown fat they have the
ability to uncouple the
proton channel
essentially transfer
protons across inner
mitochondrial membrane
similar to 24DNP
(dangerous chemical that
can kill you if you have
too much) but in a controlled way a number of organisms can do the same thing effectively
b. Proton channel also referred to as an uncoupling protein and there are a couple of types – present in brown fat, hibernating animals and human infants primarily in the upper back region
c. As scientists have done more work there is a certain amount of brown fat in adults it just not clumped
d. Deals with heat production by taking proton gradient and getting rid of it simply making heat
e. Alternate way to allow protons across the inner mitochondrial membrane that is gated by free fatty acids
f. Free fatty acid activate the thermogenin channel
g. High level of nucleotides can block the channel’s function
h. Common situation is if you have low energy and therefore high levels of ADP or GDP but usually in fat you aren’t going to be looking at high levels of fat in cytoplasm in any case i. Free fatty acid release happen sonly under conditions where triacylglycerols are hydrolyzed by enzyme triacylglycerol lipase
j. Only works in brown fat because of thermogenin(?)
K. Uncoupling in vivo – Fig. 22.47
a. Top half of figure
b. We’ve seen G
protein coupled
receptors
c. Didn’t bother to put
G protein in pic but
it’s there
d. G protein picks up
GTP after activation
interacts with
adenylate cyclase
e. Catalytic subunits of
protein kinase A is released wen 4 molecules of cyclic Amp interact with regulatory subunit dimer
f. Etc etc Activated triacylglycerol lipase
g. Heat important in hibernating animals
and infants who don’t have a lot of
insulation
h. Control of enzyme is a general control
L. Integration of Control – Fig. 22.48
a. Regardless of when you ate you have to
be making ATP
b. As long as you have oxygen you have a
source for electrons to be deposited
and a generation of proton gradient and ATP synthesis
c. If ATP concentration gets super super high or no ADP or no oxygen and NADH builds up d. High concentrations of NADH inhibit citric acid cycle in very part where NADH is a product (DH) and also at citrate synthase that controls the entry of acetyl-CoA into the cycle
M. Control of ET and OX/Phos
a. NADH/NAD+
b. ATP/ADP
c. Cytochrome c reduced/oxidized – in ETC
can control level of cytochrome C bc
ultimate place of electrons is oxygen so if
complex IV can’t put them anywhere the
cytochrome C levels build up in reduced
form
d. Control of complex IV – related to above --integrates with previous slide N. Control (cont.):
a. Control of pyruvate dehydrogenase complex
by TCA cycle activity and also control by
NADH/NAD+ ratio
b. Some positive control by calcium and energy
charge if you will so a higher ADP can push
this forward
c. Very high ATP can also inhibit
d. All control is based on what is going on in cell
in question and oxygen levels which isn’t just a
local cell issue
O. Control (cont.)
a. If citrate gets high messes with PFK
b. When talking about liver and skeletal we
didn’t talk about F26BP but not about
control with oxygen
c. In brain and heart etc where you can’t have
anaerobic run into situation where you can
have high levels of AMP which activates a
kinase (AMPK) which can phosphorylate a
specific version of the enzyme that makes
F26BP, a way to activate the glycolytic
pathway even when there isn’t enough
oxygen for the short term just to keep
going
d. AMPK is taken advantage of in cancer cells
P. Energy Yield Discussion
a. How much ATP/pair of electrons?
b. NADH ATP/2.5