BioChemistry Notes; Unit 3 (sets 14-20)
BioChemistry Notes; Unit 3 (sets 14-20) MBIOS-303
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Unit 3: Cabbage and Kings Section 3.1: Bioenergetics Living organisms are complex and ordered. Complex and ordered have low entropy and thus, requires more energy to make. The initial source of this energy on earth is mainly sunlight. Entropy: a measure of chaos/disorder. This energy cannot be reused. It is a dispersal of energy. Metabolism encompasses all the reactions in living systems. Some metabolic pathways are primarily energyproducing, catabolism (degradation). Some pathways use energy to build complex structures, anabolism (biosynthesis). A metabolic pathway is a series of reactions that can be either catabolic or anabolic in function Living systems must obey the laws of thermodynamics. Living systems can neither create nor destroy energy. They can only transform energy form one form to another. Any change that occurs must result in an increases in the entropy of the universe. Living systems utilize useable energy from the environment (e.g., from food) and release "useless" energy back to the environment (e.g., CO2, water, urea). Change in Gibbs FreeEnergy (ΔG) Consider a reaction at constant Temperature and Pressure: A + B → C + D Constant T and P covers most biological rxs. ΔG = maximum amount of useful work that can be produced from (or is required by) a process. ΔG measures how much work can be obtained (or is required) Work = moving a mass (e.g., running a marathon, pumping an ion) by a rx. ΔH = change in enthalpy. ΔH measures the difference in number of bonds and strength of those bonds between the substrates and products. Everything else being equal, ΔH is more negative when the products (compared to the substrates) have: more bonds and/or stronger bonds (e.g., C≡C > C=C > CC) T = absolute temperature Measure of change in amount of heat, Measure of intensity of heat E.g., 37 C = 310 K (add 273 to Celsius value to convert to kelvin)) ΔS = change in entropy. Form of heatenergy that cannot be reused in chemical rxs. Measure of change in chaos or disorder or randomness Everything else being equal, ΔS is more positive when there are more product molecules than substrate molecules. E.g., the rx. S → P + Q probably has a +ΔS value; X + Y → Z probably has a ΔS value ΔG = ΔH TΔSS Unit 3: Cabbage and Kings Entropy will increase with phase increase (solidliquidgas) Units Units for ΔG or ΔH are joules/mole or calories/mole Units for ΔS are in J/mol/K (extensive properties; depends on quantity) Units for temperature are in Kelvin. intensive property (doesn’t depend on quantity) Standard FreeEnergy (ΔG) Consider a reaction at constant Temperature and Pressure: A + B → C + D o ΔG = ΔG when the rx. is at "standard" conditions: Conc. of ALL the reactants (substrates AND products) are 1 M T (298 K) and P (1 atm) are also set as "standard" conditions Definitely not a reflection of reality in biological systems Tell us what the concentration at equilibrium are: ∆° = −∙ R = universal gas constant = 8.315 (joules/mol)/K Needed to convert concentrations into energy T = temperature in K (310 K = 37 C; 298 K = 25 C) RT = 2,578 at 37 ln = natural log = 2.303∙log10 C 2,478 at 25 C ∙  q /   Standard FreeEnergy with Acid/Base Consider a reaction at constant T and P: AOH + BH + H+ → CH + D+ + H2O Definition of ΔG▯ sets the conc. of ALL the reactants (including H+ and water) at 1 M, but 1 M H+ is a pH of 0 clearly of no interest for biologists Solution: "Ignore (H+)" How? Arbitrarily set the (H+) of 107 M (pH = 7) to be 1. If the pH is not 7, it is easy to mathematically correct In addition, ignore (H2O) Concentration of pure water is 55.5 M. It is definitely less in cells, but the exact conc. is essentially impossible to measure. Also "ignore" (Mg+2) not considered here Unit 3: Cabbage and Kings These "arbitrary" changes are accounted for by incorporating into the value of K'eq (compared to the value of Keq see next slide) = ∙ [+]∙ [ ���]) / ∙ [+] l = [)]∙[+] /  ∙ ∆° = −∙ lo ∆ = −∙ Note that (H ) is arbitrarily excluded, even though its concentration is 107 M; (H O) is excl2ded even 2+ though its actual concentration is up to 55.5 M; (and Mg is excluded also if necessary) Biochemists (but not chemists) refer to ΔG' as "free energy change", leaving out the word "transformed" (as we will do) + 2+ I.e., biochemists "ignore" the concentration of H , H O and2Mg ; their effects are included in the values of ΔG' Takehome lesson: Ignore the complications; use ΔG' instead of ΔG and don't worry about it lo l Relationship between ∆ and K eq o Takehome lesson: The more negative ΔG' is, the larger K'eq is What does ΔG Really Mean? A + B → C + D "ignore" the concentration of H , H O and Mg if they are involved in the reaction 2 +2 Starting with the concentration of A, B, C and D ALL being 1 M, then: ΔG tells us how much free (and useful) energy is produced (or required) when the reaction is allowed to proceed to equilibrium Unit 3: Cabbage and Kings This rarely occurs in a cell, but the value of ΔG is still necessary in order to determine how much energy a biological system can produce (or require) Analogy: how much electricity can be produced when water flows over a dam (and through turbines) into a lake (where it is at equilibrium) and the dam has a "standard" height (say 500 feet) Conditions in cells are never standard l The free energy in a cell depends on ΔG and the actual concentrations of products and reactants Massaction ration: the actual concentration ration. The actual concentrations of products and reactants are not actually at 1M. [Ex] A + B C + D Transfer of Phosphoryl group from and ATP to an acceptor ATP is very often the donor of the phosphoryl group in the biosynthesis of phosphate compounds. E.g., hexokinase Several chemical properties contribute to the high energy statues of ATP Unit 3: Cabbage and Kings Greater separation of negative charges in the products than in the substrate More favorable resonance stabilization in the products The concentration of the product H at pH 7 is very low: LeChâtelier's Principle The concentration of ATP in cells is usually greater than the equilibrium concentration, resulting in a greater value for the free energy change. Thus ATP in cells is a very potent source of chemical energy. lo ATP hydrolysis: ΔG differs form ΔG The actual freeenergy change (ΔG') of ao eaction in a cell depends upon: The standard free energy, ΔG' The ACTUAL concentrations of products and reactants (i.e., not 1 M concentrations) The freeenergy change (ΔG') becomes more favorable if: the substrate’s concentration exceeds its equilibrium concentration or the product's concentration is less than its equilibrium concentration Example: effects of actual concentration: ATP hydrolysis ΔG'⁰ = 30,500 J/mol. Using values reported for erythrocytes (in units of M): (ATP) = 2.25∙103; (ADP) = 0.25∙103; (Pi) = 1.65∙103 (T = 310 K) ∆ G =∆G °+RTln (ADP)∙(Pi) (ATP) ∙ln (0.25∙10−3 )∙1.65∙10−3 ) = 30,500 + 8.314 ∙ 310 (2.25∙10−3 ) = 30,500 + 2,578∙(8.6) = 30,500 22,000 = 52,500 J/mol Note: Cells maintain a high (ATP/(ADP) ratio that increases ΔG' considerably above ΔG'⁰ (i.e., more negative), thus providing even more "chemical driving power" The hi (ATP)/(ADP) ratio also means it takes more energy to make ATP from ADP (e.g., in oxidative phosphorylation) Unit 3: Cabbage and Kings Free energy changes are additive; Mass action ration (Q) Q (= Γ = mass action ratio) is the ratio of ACTUAL concs. of substrates and products, e.g., in a real live cell. Q can have a value greater than or less than or (rarely) equal to the value of K'eq LeChateliers Principle ATP Derived Hexokinase reaction ΔG′ = +18,500 J/mol for Glucose + Pi → Glucose6P + H2O How to convert glucose → glucose6P? Use the hienergy compound ATP ΔG′ = 52,500 J/mol for ATP + H2O → ADP + Pi Equilibrium lies FAR toward ADP production Add energy values for the two rxs.: Unit 3: Cabbage and Kings Glucose + Pi → Glucose6P + H2O +18,500 J/mol ATP + H2O → ADP + Pi 52,500 J/mol Glucose + ATP → Glucose6P + ADP 34,000 J/mol •Reaction is essentially thermodynamically irreversible! Malate to citrate in the TCA cycle K'eq for malate dehydrogenase = 6 x 106 Equilibrium FAR toward malate. How convert malate→citrate? Add energy values for the two rxs.: Malate + NAD+ = Oxaloacetate + NADH + H+ +29,700 J/mol Oxaloacetate + Acetyl CoA + H2O = Citrate + CoA + H+ 32,200 J/mol Malate + NAD+ + Acetyl CoA + H2O = Citrate + 2,500 J/mol CoA + NADH + 2 H+ K'eq for malate → citrate = 2.7 Change in Gibbs free energy ΔG < 0: rx. is thermodynamically spontaneous (= exergonic) It MAY occur!!!!!! Kinetics determines if it DOES occur!!!!!! ΔG = 0: rx. is at equilibrium (forward rate = reverse rate) ΔG > 0: rx. is thermodynamically spontaneous in the opposite direction (= endergonic) It CANNOT occur in the forward direction unless external energy is supplied happens all the time ∆=∆−∆ Example Unit 3: Cabbage and Kings Most reactions in cells are not at equilibrium ΔH = +6,007 J/mol ΔG for the forward rx. = ΔG for the reverse rx. ΔS = +22 J/mol·K T = -253K ΔG'o = 30,500 J/mol for ATP + H2O → ADP + Pi ΔG' = +30,500 J/mol for ADP + Pi → ATP + H2O -TΔS = -5,566 J/mol ΔG = +441 J/mol At temperature decreases, ΔG becomes more negative Ice stable Not all energy released during a chemical reaction is available to do useful work. Some is always lost as (mainly) heat. The amount of energy that can actually be used for work is, by definition, the free energy change, ΔG, of the reaction. Transportation of ATP, ADP, Pi, and H+ across the mitochondrial matrix ATP need to be transported out of the matrix via transport protein. This protein is an antiport protein coupling with ADP. As ATP leaves the membrane, ADP enters the membrane. There is a separate transportation protein for Pi. The translocation of Pi is symport instead of antiport. Heat Generation by Uncoupled Mitochondria Unit 3: Cabbage and Kings Important in brown adipose tissue (brown because there are so many mitochondria and red blood cells). This tissue is full of mitochondria and produce heat. Newborn humans, fawns (under hormonal control). Uncoupled ( in the brown adipose tissue) = respiration occurs but ATP synthesis does not Adipose tissue is much higher in newborns than adults Two chemical uncouplers of oxidative phosphorylation Can traverse the membrane without a transport protein, bypassing ATP synthase. This generates a lot of heat and the ATP levels decrease. The low level of ATP is fatal. Poisoning by uncouplers Symptoms include fever BOTH the acid and base forms have extensive resonance. Hence, even the charged base form is hydrophobic/lipophilic due to delocalization of the negative charge. The negative charge on the base form is so delocalized that the lipids in the membrane bilayer do not "see" it. Organic uncouplers are almost always colored due to resonance. Summary Unit 3: Cabbage and Kings Reactions can proceed only if the free energy of the products is lower than the free energy of the substrates. Reactions that require energy can proceed only if they are coupled to reactions that produce energy. Bioenergetics tells us whether a reaction MAY proceed. Kinetics tells us whether that reaction DOES proceed. Section 3.2: Membranes Membrane: Lipidbased structures that form pliable sheets. The sheets are continuous closed 3D structures composed of a variety of lipids and proteins. Carbohydrates (via glycosylation) and other components are typically also present All cells have a cell membrane that separates the cell from its surroundings. Eukaryotic cells have several types of internal membranes that separate the internal space into compartments Function of Membranes (don’t memorize) Separate the cell from its environment Allow selective import and export of metabolites and ions • E.g., import of nutrients and export of waste Retain metabolites and ions within the cell React to external signals and transmit information into the cell Provide compartmentalization within the cell, e.g. • Separate energyproducing reactions from energy consuming ones • Fatty acid betaoxidation and synthesis a good example • Separate proteolytic enzymes from crucial cellular proteins Store energy as a proton gradient • Oxidative phosphorylation is the prime example Produce and transmit nerve signals Unit 3: Cabbage and Kings Structure of Membranes (don’t memorize) Sheetlike flexible structure • ~30 Å (~3 nm) thick • Composed of two leaflets of lipids termed, the "bilayer," that is stabilized by noncovalent forces, especially hydrophobic interactions Some protein molecules span the lipid bilayer Asymmetric • Some lipids are found preferably “inside”, some "outside" • Carbohydrate moieties are always outside the cell Electrically polarized • Inside negative • –60 mV is a typical voltage gradient, but some membranes have a greater electrical gradient Fluid Mosaic Model of Membranes Integral proteins: Firmly associated with the membrane, often spanning the bilayer and held by hydrophobic interactions Peripheral proteins: Weakly associated and lie on or near one of the surfaces Lipid Bilayer of Membranes Consists of two leaflets of lipid monolayers Hydrophilic head groups interact with water Hydrophobic fatty acid tails are packed inside and are composed mainly of fatty acyl groups One leaflet faces the "outside", the other the "inside" Different membranes have specific lipid compositions The lipid composition varies in the membranes of different organisms, tissues, and organelles The lipid/protein ratio varies The type of phospholipid varies The abundance and type of sterols varies (sterols = steroids with a OH group) E.g., prokaryotes do not have sterols Cholesterol is important in the plasma membrane of animal cells, but virtually absent in mitochondria Unit 3: Cabbage and Kings Some Phospholipids Found in Membranes: Phosphatidyl The glycerol moiety is shown in red. Note the ionic hydrophilic ends compared to the longaliphatic hydrophobic ends Sterols increase membrane rigidity and permeability in many animal membranes Many cell membranes of eukaryotes contain sterols (= steroids with a hydroxyl group) Cholesterol in animals (Plants don’t contain cholesterol) Fits in between P lipid molecules, making membranes less flexible and more permeable to some molecules Precursor of steroid hormones and bile acids (digestion) Many other functions Conclusion: We need cholesterol and lots of it (but not too much) Examples of proteins in Membranes Receptors: detecting signals from outside [E.g., insulin receptor] Channels: gates, pumps for metabolites and ions [E.g. glucose transporter] Enzymes: [E.g. ATP synthase, complexes I, II, III, IV] Unit 3: Cabbage and Kings ~30 Å Lipids will often form alpha helix to increase number of H bonds in polypeptide backbone 1.5 Å per amino acid in an alpha helix (= the "rise") ~20 amino acids per ~30 Å across the lipid layer of a membrane. Peripheral membrane proteins: Associate with the polar head groups of membranes, interacting with the hydrophilic lipid heads Integral membrane proteins: Span the entire membrane. Hydrophobic segments in the transmembrane protein interact with the hydrophobic regions of the membrane. Most common: 1, 7 or 12transmembrane segments Ex: bacteriorhodopsin: a 7transmembrane protein Major Types of Transport Systems Uniport: a substrate is transported from one side of the membrane to the other Symport: two or more substrates need to be transported together across a membrane in the same direction Antiport: as one substrate crosses the membrane, another membrane need to be transported across the membrane in the opposite direction Oxidative phosphorylation (ATP and ADP in and out of matrix. Pi and H+ in and out of matrix) Section 3.3: Gluconeogenesis The synthesis of new glucose Glucose is a major fuel in most cells Produces energy upon degradation Can be stored in the form of a polymer Glycogen in animals, starch or sucrose in plants Unit 3: Cabbage and Kings Glucose is a precursor of: Many amino acids, Some lipids, (Deoxy) nucleotides in DNA and RNA, Cofactors of many enzymes, Etc. Source of energy in muscle for bursts of contractions Fatty acids, blood glucose CO2 rest/light activity (aerobic metabolism) Phosphocreatine, muscle glycogen Creatine, Lactate bursts of heavy activity (anaerobic metabolism) Lactate Dehydrogenase Converted to glucose if it need to be used Converted to Glycogen if it needs to be stored Cori Cycle Vigorously active muscle generates lactate from glucose via glycolysis. Lactate enters the blood and is transported to the liver. In liver, lactate is converted to glucose via gluconeogenesis (and much of this glucose is stored as glycogen, a polymer of glucose). Unit 3: Cabbage and Kings Gluconeogenesis (anabolic/biosynthetic pathway) Unit 3: Cabbage and Kings Glycolysis vs Gluconeogenesis The 2 pathways operate in opposite directions Glucose is the starting compound for glycolysis and the end product of gluconeogenesis The two pathways share each of the 7 thermodynamically reversible reactions Each of the 3 irreversible reactions of glycolysis must be somehow reversed in gluconeogenesis This is accomplished by using completely different reactions (and thus enzymes) at each of the 3 nonshared steps Pyruvate Kinase in glycolysis: Pyruvate Carboxylase in gluconeogenesis: (in matrix) 4 hienergy bonds; 1 in PEP, 1 in ADP, 2 in ATP Unit 3: Cabbage and Kings Oxaloacetate in moved from matrix to Cytosol via transport system: PEP Carboxykinase: Requires two energyconsuming reactions separated by a transport step 1st rx.: pyruvate carboxylase converts pyruvate → oxaloacetate in the mitochondrial matrix o Carboxylation using ATP as energy source to make the CC bond (the vitamin biotin is a cofactor) Oxaloacetate is transported from the mitochondrial matrix into the cytosol via malate Second rx.: phosphoenolpyruvate carboxykinase converts oxaloacetate → PEP in the cytosol o Phosphorylation by GTP and decarboxylation Two additional steps with different enzymes Catalyze reactions in opposing steps of glycolysis vs. gluconeogenesis o Fructose 1,6bisphosphatase: Fructose 1,6bisphosphate + H2O ▯ Fructose 6Phosphate + Pi Reverses phosphofructokinase step in glycolysis o Glucose 6phosphatase Unit 3: Cabbage and Kings Glucose 6phosphate + H2O ▯ Glucose + Pi o Reverses hexokinase step in glycolysis These two gluconeogenic rxs. are also irreversible Note that, for each of these two steps, a kinase adds a phosphoryl group in glycolysis and a phosphatase removes the phosphoryl group in gluconeogenesis Phosphofructokinase in Glycolysis: Fructose 1,6bisphosphatase in Gluconeogenesis Hexokinase in Glycolysis: Glucose 6phosphatase in Gluconeogenesis: Gluconeogenesis enzymes only found in liver or kidney Gluconeogenesis is energetically expensivebut worth it 2 Pyruvate + 4 ATP + 2 GTP + 2 NADH + 2 H + 4 H O 2 Glucose + 4 ADP + 2 GDP + 6 Pi + 2 NAD + Costs 4 ATP, 2 GTP, and 2 NADH Brain, the nervous system, and erythrocytes can use only glucose as fuel under "normal" conditions When glycogen stores are depleted or not available, we need to make glucose ourselves E.g., starvation, diabetes, low carbs diet Precursors for gluconeogenesis Animals can produce glucose from many small molecules Unit 3: Cabbage and Kings Pyruvate, oxaloacetate and metabolites that can be converted to TCAcycle or glycolytic intermediates [E.g., lactate, glycerolP] Most amino acids (termed glucogenic amino acids) o These amino acids are derived mainly from proteolysis (protein degradation to a.a.) Animals cannot convert fatty acids to glucose Product of betaoxidation is acetyl CoA Cannot convert C2 acetyl CoA to C3 pyruvate or C4 oxaloacetate Plants, yeast, and many bacteria can do this, thus they can produce glucose from fatty acids The 18 Glucogenic amino acids A, C, D, E, F, G, H, I, M, N, P, Q, R, S, T, V, Y, W Only K and L cannot be converted to glucose Takehome lesson: Protein degradation (proteolysis) can provide precursors for glucose biosynthesis. Especially important during starvation Summary of Gluconeogenesis Glucose catabolism is absolutely necessary for survival by animals Sometimes sufficient glucose is not available for degradation A common example is starvation (or diabetes, which is a form of starvation) Under conditions of lack of glucose availability, animals can synthesize glucose from other metabolites, including most amino acids This pathway is gluconeogenesis Gluconeogenesis is "almost" the reversal of glycolysis Three steps differ, each one being irreversible in the direction it proceeds Source of Energy in Brain NorStarvation (more than 12 Nuthours) Units 3: Cabbage and Kings Section 3.4: Enzyme Kinetics II MichaelisMenten kinetics: LineweaverBurk plots LB equation converts MM hyperbola into a straight line Inverse of a hyperbola is a straight line Straight lines are easier for humans to interpret than hyperbolas Generalization: Equation for a hyperbola: Inverse of a hyperbola Plot of 1/x vs. 1/y is a straight line with a Yintercept of c/a and a slope of b/a Apply this to the MM equation (next slide) Units 3: Cabbage and Kings The intercept = 1/V max Slope = K /M max Enzyme Inhibition Irreversible inhibitors usually form a covalent bond with the enzyme and hence stay bound to the enzyme One inhibitor molecule can permanently shut down an enzyme molecule Often either toxins or pharmaceutical drugs Reversible inhibitors bind to and dissociate from the enzyme Often structural analogs of substrates or products Often used as drugs to slow down a specific enzyme A reversible inhibitor can bind to the: free enzyme and prevent binding of the substrate and/or enzymesubstrate complex to prevent formation of product Units 3: Cabbage and Kings MM plot vs. LB plot Ki = how tightly the inhibitor binds. The smaller Ki is, the more tightly the inhibitor binds Units 3: Cabbage and Kings Comments: Competitive Inhibition Competes with substrate for binding to the enzyme Both bind at the active site Does not affect catalysis, only binding of substrate to the enzyme No change in Vmax; apparent increase in KM Its LineweaverBurk plot lines intersect at the yaxis Reminder of what we have already seen: CO and oxygen both binding to iron in hemoglobin Antidote for ethyleneglycol poisoning Synthesis of Cholesterol (first step) Medication for high cholesterol utilize competitive inhibition MM model of 3 types of reversible inhibitors Units 3: Cabbage and Kings An enzyme inhibitor can be characterized by its 2 dissociation constants, Ki and Ki': Competitive: K'i = ∞ (i.e., no ESI forms) Uncompetitive: Ki = ∞ (i.e., no EI forms) Mixed: Ki ≠ K'i o Noncompetitive: Ki = K'i (special case of mixed inhibition, rare in Nature) Mixed Inhibition: Substrate and inhibitor bind to different sites •Binds either free enzyme or enzymesubstrate complex •Binds to the regulatory site •Inhibits both substrate binding and catalysis •Results in a decrease in Vmax and an apparent increase in KM •Its LineweaverBurk lines intersect left of the yaxis Units 3: Cabbage and Kings Increasing Km means it takes more substrate to get to some velocity when mixed inhibition occurs Uncompetitive Inhibition: Uncompetitive inhibitors bind at a site different from the substratebinding site, but bind only to the ES complex Inhibitor can only bind after substrate is bound Only binds to ES complex Does not affect substrate binding Inhibits catalytic function Results in a decrease of Vmax and an apparent decrease of KM Units 3: Cabbage and Kings Does not change KM/Vmax Its LineweaverBurk lines are parallel Example: Roundup (glyphosate) is an inhibitor of EPSP synthase (an enzyme essential to plant growth). Roundup is used to kill weeds. It is a uncompetitive inhibitor of shikimate3 P but a competitive inhibitor of PEP Final Comments on MM Kinetics Mixed and uncompetitive inhibitions occur essentially only with enzymes that have 2 substrates Mixed inhibition indicates one type of 2substrate catalytic mechanism, uncompetitive inhibition another mechanism Many practical inhibitions are irreversible E.g., organofluorophosphates and enzymes with the catalytic triad (one example: sarin and acetylcholinesterase) Units 3: Cabbage and Kings Acetyl Cholinesterase Organofluorophosphates inhibit cholinesterase which inactivates the neurotransmitter acetylcholine This esterase has the same catalytic mechanism as serine proteases Nerve poison (Sarin) used by Sadam Hussein, Aum Shinrikyo and Bashar alAssad Heavy Metal Poisoning EnzSH + M+ → EnzSM + H+ E.g.: Pb, Hg, Cd, Ag, etc. o Can bind any available sulfhydryl group (on Cys residues) o May or may not be near the active site o Erythrocytes and the central nervous system are especially vulnerable o Essentially irreversible inhibition E.g., major site of lead poisoning: porphobilinogen synthase o Early step in biosynthesis of heme o Leads to anemia Assumption vs. Reality Although some examples exist of true uncompetitive and noncompetitive inhibitors, in most cases, the kinetics are not that simple. Uncompetitive inhibition: inhibitor binding should occur only if the active site is occupied by substrate. But in most cases, the inhibitor will have some affinity for the unoccupied enzyme as well. Noncompetitive inhibition: the inhibitor affinity should be unchanged regardless of whether substrate is bound or not. In fact, the affinity for the inhibitor usually changes when substrate is bound. True competitive inhibition is common. In reality, we see competitive and mixed inhibition only. Inhibition Spectrum: No ESI No EI Units 3: Cabbage and Kings Enzyme Regulation via… noncovalent modification; often readily reversible covalent modification; often irreversible unless another enzyme becomes involved Units 3: Cabbage and Kings Inhibition Further Explained Section 3.5: Fatty Acid Synthesis Catabolism and anabolism of fatty acids occur via different pathways in different subcellular locations Catabolism of fatty acids (betaoxidation) produces: Acetyl CoA and Reducing power (NADH and FADH2) Location: mitochondria Anabolism of fatty acids requires Acetyl CoA and malonyl CoA and reducing power from NADPH (not FADH2) Location in animals: cytosol in animals Units 3: Cabbage and Kings Preview of Fatty Acid Synthesis Fatty acids are built in several cycles, adding one acetate unit at a time The acetate unit comes from activated malonate in the form of malonyl CoA Each cycle involves reduction of a carbonyl carbon to a methylene carbon, which requires 2 x 2 electrons Acetyl coenzyme A carboxylase: (Synthesis of malonyl CoA from acetyl CoA) Fatty Acid Synthase: Catalyzes a repeating 4step sequence that elongates the fatty acyl chain by two carbons at each step NADPH is the electron donor Two enzyme bound SH groups are activating groups FAS I in vertebrates and fungi FAS II in plants and bacteria In general, NAD and NADH are involved in catabolic reactions while NADP and NADPH are involved in biosynthetic reaction (all are pyridine nucleotides) Fatty Acid Synthase Type I: Animal and Fungi Occurs on a single polypeptide chain in vertebrates Only product: palmitate 16:0 Units 3: Cabbage and Kings C15 and C16 derived the first acetyl CoA, the other C atoms from malonyl CoA All of the enzyme active sites are on one protein FAS II in plants Summary of Fatty Acid Synthesis: Overall strategy: transfer an acetyl unit (2 C atoms) from malonylCoA to a growing chain and then reduce it Reaction involves cycles of four enzymecatalyzed steps Condensation of the growing chain with activated acetate Reduction of a carbonyl to an hydroxyl Dehydration of an alcohol to a transalkene Reduction of an alkene to an alkane The growing chain is initially attached to a cysteine residue in the enzyme via a thioester linkage During condensation, the growing chain is transferred to a S atom in the acyl carrier protein After the second reduction step, the elongated chain is transferred back to a cysteine in the fatty acid synthase The saturated C16 fatty acid (palmitic acid) is the end product in the mammalian system Acyl Carrier Protein (ACP): Contains a covalently attached prosthetic group 4’phosphopantetheine Is a long flexible arm that carries intermediates from one enzyme active site to the next one Is a separate protein from the fattyacidsynthase protein, although they are linked to one another Delivers an acetyl group (in the first cycle) or a malonyl group (in all subsequent cycles) to the fatty acid synthase Shuttles the growing chain from one active site to another during the fourstep reaction in a cycle Pantothenic acid is a B vitamin that also a CoA Fatty Acid Synthesis I Units 3: Cabbage and Kings Start with 2 carbons end up with 16 Fatty Acid Synthesis II NADPH is the reducing agent Fatty Acid Synthesis III Repeat 7 times to make 16 carbon chain Units 3: Cabbage and Kings Fatty Acid Synthesis IV Enzymes in Fatty Acid Synthase (don’t memorize) Chain transfer/charging: ACPSacetyltransferase Chain transfer/charging: ACPSmalonyltransferase Condensation with an acyl group: ketoacylACP synthase Reduction of carbonyl to hydroxyl: ketoacylACP reductase Dehydration of alcohol to alkene: hydroxyacylACP dehydratase Reduction of alkene to alkane: EnoylACP reductase Release of final fattyacid product: PalmitoylACP hydrolase Summary The intermediate product after the first cycle is butyrylACP Bound to the SH group of phosphopantetheine The butyryl group is transferred to the SH group of a Cys in ketoacylACP synthase In the first cycle, acetyl CoA was bound here A new malonyl CoA binds to ACP After the next cycle of four steps, the 6C acyl group is the intermediate product bound to ACP Etc., etc. ...... Stoichiometry of Fatty Acid Synthase I 7 acetyl CoAs are carboxylated to make 7 malonyl CoAs 7 Acetyl CoA + 7 CO2 + 7 ATP ▯ 7 Malonyl CoA + 7 ADP + 7 Pi Then 7 cycles of condensation, reduction, dehydration and reduction + Acetyl CoA + 7Malonyl CoA + 14NADPH + 14H Palmitate (16:0) + 7CO 2+ 8CoA + 14NADP+ + 6H O 2 Reminder: 7 cycles to make C16 (analogous to betaoxidation) Units 3: Cabbage and Kings Comments on Fatty Acid Synthase I An acetyl group (from acetyl CoA) is the first group to bind It binds to the S atom of the 4'phosphopantotheine segment of acyl carrier protein (ACP) In order to make room for a malonyl group, the acetyl group is transfered to the S atom of a cysteine Malonyl CoA (at each cycle) enters by attaching to the S atomof ACP The alphaC of malonylACP attacks the alphaC of the acetyl group CO2 from the malonyl group is released and a newly extended betaketoacylACP is produced While remaining attached to ACP, the betaketoacyl group is: reduced to an alcohol then dehydrated to a CC double bond then reduced at the double bond to make a saturated fattyacyl group still attached to ACP In order to make room for the next malonyl group, the saturated fattyacyl group is transfered to the S atom of a cysteine Malonyl CoA enters by attaching to the S atom of ACP Repeat, repeat, etc. When the fattyacyl group reaches 16 C atoms, it is hydrolyzed to palmitate, the final (and sole) product C15 and C16 are derived directly from acetyl CoA The other C atoms are derived directly from malonyl CoA The S atom of the cysteine serves as a "temporary" dock for the growing saturated fattyacyl group Before it is transfered to the alphaC of malonyl CoA The S atom of the ACP is where all the action is Fatty acid synthesis occurs where NADPH levels are elevated Units 3: Cabbage and Kings Unit 3: Cabbage and Kings Section 3.6: Metabolic Regulation Homeostasis: biological conditions remain stable and relatively constant Organisms maintain homeostasis by keeping the concentrations of most metabolites mostly constant In a steady state, the rate of synthesis of a metabolite equals its rate of degradation Pathways are at a steady state unless perturbed (e.g., by changing environmental conditions) After perturbation a new steady state will be established Homeostasis depends on metabolic regulation The flow of metabolites through the pathways is regulated to maintain homeostasis Sometimes, the levels of required metabolites must be altered very rapidly, sometimes quite slowly Feedback Inhibition Often the last product of an anabolic pathway inhibits its own biosynthetic pathway Often it is the first unique enzyme in the pathway that is the one inhibited E.g., isoleucine inhibits threonine deaminase, the first unique enzyme in the isoleucine biosynthetic pathway F inhibits AB … Usually Rates of Metabolic reactions depend on many factors Concentration of the enzyme Rate of synthesis of the enzyme vs. rate of its degradation Catalytic activity of the enzyme (turnover number) Concentrations of substrates and products Concentrations of effectors o activators (+ effector) or inhibitors ( effector) Covalent modification of the enzyme molecule o pH, ionic environment o Temperature o Location in the cell Reminder: Total enzyme activity = # of enzyme molecules x (catalytic activit/ enzyme molecule) Unit 3: Cabbage and Kings Regulation of metabolism by the cellular levels of ATP and AMP E is for energy (ATP) (Hi ATP) indicates energy sufficiency or excess: anabolism favored Hi AMP (and usually ADP) indicates energy dearth: catabolism favored The (ATP) / (AMP) ratio is a major control parameter in cells since the flux through so many energy requiring and energyproducing reactions is affected Side remark: Pi = inorganic phosphate = mix of HPO4= + H2PO4ˉ Why make a pathway sensitive to the levels of ATP and AMP? (Fact: ADP is rarely used as an effector.) Highly controlled reactions in TCA: 1) Acetyl CoA Citrate 2) Isocitrateaketoglutarate 3) aketoglutarate Succinyl CoA 1) Citrate Synthase: inhibited by ATP and NADH b/c a Pi needs to be releases from Acetyl CoA. (Needs ADP to donate a Pi group to) 2) Isocitrate dehydrogenase: inhibited by ATP for the same reason. ADP will stimulate this reaction as it did in citrate synthase 3) aketoglutarate dehydrogenase: inhibited by NADH because the a proton needs to be released but NADH cannot accept protons. Needs NAD to proceed Unit 3: Cabbage and Kings Section 3.7: Saturday Night Craziness First two steps of alcohol metabolism (mostly in the liver): Most metabolic effects of overconsumption of alcohol are explained by excess production of NAHD There are three sources of blood glucose o Diet (intestine) o Gluconeogenesis o Glycogen (stored in the liver) Alcohol can lead to hypoglycemia the source of glucose from the diet is low when you don’t eat before drinking this depletes the glycogen reservoirs as well gluconeogenesis is the only sources of glucose excess NADH. Pyruvate will be reduced to lactase and pyruvate and Oxaloacetate concentration will decease o gluconeogenesis compromised Unit 3: Cabbage and Kings o central nervous system starved