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LEHIGH / OTHER / CHMBIO 372 / How can we control so many processes happening in the cytoplasm?

How can we control so many processes happening in the cytoplasm?

How can we control so many processes happening in the cytoplasm?



How can we control so many processes happening in cytoplasm?

Elements of Biochemistry II

Week 3


Succinate Dehydrogenase (Extra material) – Page 811

∙ Succinate to fumarate

∙ Fumarate is always a trans double bond

∙ The enzyme is able to specifically abstract the electrons and protons from opposite faces in a way  that you always get the trans product

∙ The trans aspect is one of the things you want to keep in mind for structures****

A. (J.) Fumerase – Fig. 21.24

a. ~

B. (K.) Malate Dehydrogenase – Page 813

a. (S)-Malate to Oxaloacetate

b. Looking at the oxidation of the carbon

What is the hormones on muscle?

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 Don't forget about the age old question of What is the classification of human societies?

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

Citric Acid Cycle Summary

∙ Acetyl CoA + 3 NAD+ + FAD + Pi + GDP -> 2CO2 + CoASH + 3 NADH + FADH2 + GTP ∙ So from glucose the energy yield is: 2 ATP + 2 NADH  

What is the energy differences?

∙ Through PDH the yield is: 1 NADH/pyruvate

∙ And through TCA the yield is: 3 NADH + FADH2 + GTP/AcetylCoA

∙ All is net energy recovery from the oxidation reactions

∙ Actually getting a lot of yield from energy

∙ 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 ∙ For now, just know yields of reduced electron carriers and GTP & ATP for each portion of the  scheme If you want to learn more check out What are the primary forms of business organization?

Discussion on Isotopes

∙ Q: Remember the prochiral discussion? Where would the triangle and star be on a-ketoglutarate? ∙ Star is on CH3’s on pyruvate and ACoA and the triangles are on the carbons next to those ∙ On citrate the star is on the carbon 1 down from the triangle (Fig. 21.1)

∙ A: The triangle is on the top carbon, the star is right underneath.

∙ Q: What about on oxaloacetate?

∙ 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.

∙ 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?

∙ Isotope chasing:

∙ First time around w/ newly added ACoA, released as free carbon dioxide

∙ Starred carbon from a-ketoglutarate is released as CO2 etc. The CO2 released in the step before  that is also from oxaloacetate We also discuss several other topics like Define puberty.

∙ Should help you learn the TCA cycle better too and what is actually happening

Regulation – Fig. 21.25

∙ Overall perspective: intracellular considerations, no significant hormonal control ∙ A-ketoglutarate dehydroenae is essentially the same E3 as pyruvate dehydrogenase that a ketoglutarate dehydrogenase, reaction at bottom left is similarly regulated,  

∙ High concentrations of calcium activate

∙ High levels of NADH negatively regulate isocitrate dehydrogenase

∙ ATP also negatively regulates isocitrate dehydrogenase

∙ Look at diagram to find negative regulation

∙ 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

∙ Make or eliminate Citric acid cycle intermediates

∙ Just said excess lactate makes way to liver and in liver is converted to glucose ∙ 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 If you want to learn more check out What is another reasonable explanation for an increase in autism rates?

∙ 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

∙ This particular reaction, if using TCA intermediates to make glucose, will require us to make new  TCA intermediates

∙ There are other reasons why you would need other TCA cycle intermediates ∙ 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 If you want to learn more check out Who was martin luther?

∙ Basically, when we consume protein we have way more AA than we need

∙ 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 ∙ Worth recognizing the fact that a lot of input in the TCA cycle is through AA ∙ Can also use the TCA intermediates to reform AA

∙ Last use is the use of citrate for fatty acids and cholesterol

∙ 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

∙ The 4 carbon part of the citrate gets shoved back into cytoplasm and gets reused ∙ Citrate is essentially a carrier to get ACoA into cytoplasm where __ is produced ∙ Constantly in flux, a significant thing it’s doing is giving us energy from foods we consume ∙ Also heavily involved in some other activities Don't forget about the age old question of Who discovered the surface structures of the moon?

∙ What happens when we have low TCA cycle intermediates and have a need to run the TCA at a  more rapid rate?

Oxaloacetate from Pyruvate

∙ Anaplerotic

∙ 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

∙ Its possible that what you need to do is store some ACoA, but need to attach it to citrate to shove it  into cytoplasm

∙ So high levels of ACoA shows we don’t have enough of the intermediates

∙ So we has pyruvate carboxylase, activated by high levels of ACoA, provides a way to make  oxaloacetate from pyruvate and ATP and CO2

∙ 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

∙ 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

∙ 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

∙ This reaction and its control turn out to be a crucial mechanism

∙ 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 nonreducing, addition and removal of glucose happens at the nonreducing 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

vii. 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 UTP  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. 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. Unk

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 excerted muscles. Had to turn on glycogen__. 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 – 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. Unk

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 muslces

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. Subinit GM (m=muscle) and a subunits of PP1c associated with glycogen iv. Role of subunit is to put the phoisphatase where it needs to be to function,  associating with with glycogen so it can have activity

v. When its associated with the singly phosphorylated it is most active, with two  phos. 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 phos.

viii. Its pretty easy to understand why we would want the event to happen (double  phos) because we might not want it running all the time

ix. Two phos means enzyme is not localized in an acive 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 phos of enzymes from before and  partially by blocking activity of this phosphatase so that the phos groups stay on xi. EX: suppose you were in UC eating jelly donuts instead, level of blood glucose  high. Leads to insulin being released. Insulin causes a single phosphorylation  event on GM1 which hmeans any available PP1 gloms onto GM and is  associated with glycogen and can remove a phos and start process of turning  system around.

xii. ^Part of the reason why this works well is that ina resting state, the most active  version of the phosphatase is the one associated with a singly phos 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 phos but small molecules  help as well.

xiii. Free glucose can force T state of molecule before. By pushing glycogen phosphorylase into T form, phos 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 phos 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 store as glycogen

ii. Glucose is degraded and fatty acids are made (F-2,6-P is made to activate  glycolysis)


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