ENZYMES: PROTEINS THAT ARE CATALYSTS• I. Catalytic Power- ability to increase reaction rates• A. Most reactions in biological systems occur slowly in the

absence of catalysts; for practical purposes the rate is zero. Reactions are not at equilibrium except as enzymes move them toward EQ.

• B. Fast enzyme? catalase: each molecule catalyzes 10,000,000 times per second

• C. Rate may be > 107 X as fast as uncatalyzed.

Specificity toward substrates (reactants)• A given enzyme will catalyze

a single reaction (or, in some cases, a single type of reaction). To be so specific, it must "recognize" its substrates and not interact strongly with the other molecules it encounters. The specificity is determined by complimentary 3-D shapecomplimentary 3-D shape (or “fit”: square peg can't fit round hole) ANDAND reversible reversible interactions.interactions. (fig 13-1, p460)

Substrate Specificity• Examples:• 1. There are a variety of

proteolytic enzymes, which catalyze hydrolysis of peptide bonds of other proteins (as in digestion of food). Some of these will cleave bonds only on a particular side of a particular type of AA residue, others between two particular residues.

• 2. Most enzymes that act on AAs are specific for the L isomer. Beyond this, enzymes are often stereospecific in that they will specifically remove one of 2 "equivalent" groups from a symmetric molecule.

Regulation: Control of Enzyme Activity• If all molecules of a given enzyme are inactivated, reaction If all molecules of a given enzyme are inactivated, reaction

rate approaches zero. If all are activated (or not rate approaches zero. If all are activated (or not deactivated), reaction proceeds toward EQ at high rate.deactivated), reaction proceeds toward EQ at high rate. We shall see that usually control of one reaction in a pathway results in control of all of them.

• Types: • 1. AllostericAllosteric: • a. as with Hb: small indicator molecules • b. Regulatory proteins are themselves regulated and

then act to control other proteins.• 2. Covalent modificationCovalent modification: often a hydroxyl containing residue

(ser, thr, tyr) will be phosphorylated at its –OH group. This results in stabilization of one form of the protein, as in CO2 effect on Hb.

• 3. Proteolytic activation: the enzyme is synthesized in inactive form, becomes active after a segment of its polypeptide chain is removed. (common for food digesting proteases)

• Enzymes interconvert forms of energy.

• Living things are "chemical engines".

• Foods are energy fuels; the energy released when they are metabolized is used as mechanical (muscle contraction), osmotic (and nutrient transport), chemical (biosynthesis), etc. energy.

Energy: Thermodynamics• A. Second Law: For all spontaneous processes,

the entropy of the universe increases. In biosynthetic processes the entropy decreases, but this is offset by a larger increase in the entropy of surroundings. (ΔS (universe) = ΔS (system) + ΔS (surroundings) )

• B. The above statement of the Second Law is difficult to use practically:

• 1. It can be converted mathematically to the following result: a reaction occurs a reaction occurs spontaneously only if ΔG < 0. It is spontaneously only if ΔG < 0. It is atat equilibrium if equilibrium if ΔG = 0ΔG = 0. (ΔG is the "free energy change") (spontaneously means left to right. We say the reaction is not spontaneous if ΔG > 0, but what this means is that the reaction proceeds from right to left as written.)

Energy: Thermodynamics• 2. We will often be concerned with readily reversible reactions.

The reaction proceeds at a significant rateat a significant rate from right to left andand from left to right:

• at equilibrium the rates of the reactions in each direction are equal and ΔG = 0.

• If ΔG < 0 the rate of the reaction to the right is greater than If ΔG < 0 the rate of the reaction to the right is greater than that to the left and we say the (that to the left and we say the (netnet) “) “reaction goes to the reaction goes to the right”right” (until EQ is reached). (until EQ is reached).

• If ΔG > 0 we say the (net) “reaction goes to the left”. • Note: Spontaneously means "without proportional input of

energy". It does It does notnot mean "occurs immediately" or "occurs mean "occurs immediately" or "occurs rapidly".rapidly". There is no relationship between ΔG and rate.

• 3. ΔG is independent of path. The ΔG for glucose oxidation is the same whether the glucose is burned in air (a reaction in which oxygen molecules collide with glucose molecules) or occurs in a living system without flame or any direct contact between oxygen and glucose.

Thermodynamics and Equilibrium• For the reaction: aA + bB ---> cC + dD; • ΔG = ΔGΔG = ΔGoo' + 2.303 RT log Q; where ' + 2.303 RT log Q; where • Q = [C]Q = [C]cc[D][D]dd/[A]/[A]aa[B][B]bb at the existing conditionsat the existing conditions and and• ΔGΔGoo' = -2.303 RT log Keq. ' = -2.303 RT log Keq. • The superscripts on ΔGsuperscripts on ΔGoo' indicate ' indicate standard conditionsstandard conditions,,

namely [A] = [B] = [C] = [D] = 1 namely [A] = [B] = [C] = [D] = 1 MM ( (ifif A, B, C, and/or D are A, B, C, and/or D are aqueous substances) (and aqueous substances) (and ifif A, B, C, or D is a gas, P A, B, C, or D is a gas, PAA =P =PBB = = PPCC = P = PDD = 1 atm for a gas), = 1 atm for a gas), and the prime indicates a special standard condition for biochemistry: pH = 7.pH = 7.

• The value of ΔGo' indicates the spontaneous direction of the reaction under standard conditions as for ΔG above.

• Note that to calculate ΔGo' we must first calculate Keq from the EQ concentrations of A, B, C, and D.

• If the system is at EQ: ΔG = 0 and Q = Keq. • (Note: R= 8.31 J/molK = 8.31X 10-3 kJ/molK and 2.303RT = 5.7 5.7

kJkJ at 298K.)

Thermodynamics and Equilibrium• 1. Definition: "position of

equilibrium" is the relative amounts of reactants and products at EQ.

• a. If, at EQ, the concentrations of C and D are greater than those of A and B (actually [C][D] > [A][B]) then "products are favored", Keq>1 and ΔGo' <0.

• b. If K<1, log K<0, ΔGo' >0, and "reactants are favored" ([C][D] < [A][B]).

• c. ΔG = -5.7 kJ if Keq = 10 and log Keq=1. For each 5.7kJ change in ΔGo', the Keq changes by a factor of 10. see table 3-3, p49

Thermodynamics and Equilibrium

• 2. Note that the spontaneity of a reaction depends on the 2 factors in the ΔG expression:

i. ΔGo' indicates the characteristics of the substances in the reaction regarding which are more stable and favored at equilibrium.

• ii.Q indicates existing concentrations. • If Q=10 and Keq=3 then there are more products

(and/or less reactants) than at EQ and the reaction will go left even though products are favored.

Kinetics: effects of enzymes on reaction rates

• 1. Enzymes have no effect on ΔGo', ΔG, Keq, or the position of EQ.

• 2. Enzymes cannot make a reaction Enzymes cannot make a reaction go in a go in a certain directioncertain direction, they only make it go , they only make it go faster faster in the directionin the direction (net) specified by ΔG. (net) specified by ΔG.

• 3. Enzymes actually accelerate the rate in 3. Enzymes actually accelerate the rate in both directions equallyboth directions equally (by an equal (by an equal factorfactor). ).

• 4. They increase the rate at which the 4. They increase the rate at which the reaction goes toward EQ.reaction goes toward EQ. Most biological reactions are not at EQ in a living cell.

How Do Enzymes Achieve Catalysis?

• Enzymes accelerate the reaction by lowering lowering the activation energythe activation energy of the reaction. They do this by stabilizing the transition statestabilizing the transition state.

• 1. What does this mean? And how do enzymes do it?

• By forming additional bondsforming additional bonds with the transition state that they don't have with reactants or products. (R, P, and TS have many otherother bonds that are the same for all three.)

• a. Recall that bond formation releases energybond formation releases energy, leaving the bonded substances at a lower bonded substances at a lower (more stable) energy(more stable) energy.

b. Example: tyrosyl-amino acyl tRNA synthetase.

• i. In this reaction, the carboxyl O of tyrosine is to become linked to the first phosphate of ATP (adenosine triphosphate) with the other two phosphates being released.

• ii. The tyr and nonphosphate portion of ATP are bonded to the enzyme throughout the reaction. But the third phosphate of ATP is bonded only in the transition state.

• iii. The geometry at P of the first phosphate is tetravalent (tetrahedral) in reactants and products, but it's pentavalent (trigonal bipyramidal) in the transition state.

• iiii. When the geometry is pentavalent, the end phosphate is placed where it can H-bond to two of the enzymes side chains at the active site. This is the only time these bonds are made.

• 2. This is called specific binding of the transition state.

Molecular Mechanism of Allosteric Regulation of Enzyme Activity

• (Covalent modification by phosphorylation of side chain OH groups results in similar effects)

• A. Example Enzyme (E): Aspartate Transcarbamoylase (ATCase)

• 1. Catalyzes the first reaction in pyrimidine nucleotide synthesis, linking the substrates aspartate (asp) and carbamoyl phosphate (CP). (Note that ring closing by formation of an amide bond gives a pyrimidine: 2,4 diketo, 6 carboxyl)

• 2. Regulatory effectors:

• a. inhibited by CTP. Metabolic Relationship (MR) of CTP to E: indirect indirect productproduct - CTP is the product of the last step in this pathway. (Indirect feedback inhibition)

• b. activated by ATP. MR: ATP and CTP are used (along with UTP and GTP) in RNA synthesis

Metabolic Logic of Regulatory Effects

• a. When [CTP] is high, ATCase activity to produce more is not needed. Inhibition by CTP conserves substrates for other uses.

• b. ATP and CTP are used in RNA synthesis (along with GTP and UTP). When [ATP] is high, RNA synthesis is well supplied ONLY IF [CTP] is also high. Activation of E by ATP coordinates nucleotide synthesis/supply.

Kinetics of ATCase• Kinetics of E: (rate(v) vs. [asp]) • The curve (Fig 13-5) indicates positive cooperativity,

as with Hb. The affinity for asp is low at low [asp], and is higher at higher [asp], where several molecules of asp are bound.

• Subunit composition: c6r6. • The 6 identical catalytic (c) subunits are arranged as two

trimers (one trimer red, one blue in Fig 13-7). • When separated from the r subunits, the c’s bind asp and CP

and are active but the kinetics are NOT cooperative. The separated c’s do not bind CTP or ATP, are not affect by them.

• The regulatory (r)subunits are three dimers (green in Fig 13-7), which link the c trimers to each other. They bind CTP and ATP but have no catalytic activity.

Molecular Events: Each of the six catalytic subunits has two domains; one binds asp and the other, CP. When substrates bind (pairs of CP + asp) in the t/T form, asp and CP are distant and can’t make contact. But, a shift in those subunit(s) occurs in which the domains move to bring CP and asp together (tertiary structure: t r). This causes the subunits to move away from each other (T--->R) so that t r is not blocked by the asp domain on the subunit across from it.

The catalytic trimers move away from each other and rotate in relation to each other. More intersubunit interactions are broken than are formed.Notice how the catalytic subunits in red and blue have been able to rotate (t r to bring asp and CP together) in the R form on the right and how these subunits block each other in the T form on the left.

• Summary of changes in interactions and energy (ΔH and so ΔG) which explain kinetic properties (affinity/cooperativity) of ATCase:

• 1. T <--->R When no substrates are bound the T form is favored. There is a net decrease in intersubunit (IS) interactions when T--->R (intersubunit bonds broken) ΔHIS is a large positive (and ΔG is more positive) for T---> R.

• 2. T + asp + CP---> asp-R-CP (1st, 2nd, etc asp, CP bind) There is a gain of interactions between E and substrates and between substrates, ΔHSB for substrate binding (SB) is a large negative. The sum ΔGSB + ΔGIS = small (-), low affinity (or at least low rate)

• 3. asp-R-CP + n CP + n asp---> nasp-R-nCP where n=2, 3, 4,5 or 6. The IS interactions are already broken, so ΔG = n ΔGSB (and no ΔGIS) so ΔG is large (-) and affinity of R form is high

• 4. Activation by ATP, which binds strongly only to R form, keeps more E in R form.

• R-nATP ---> T + nATP breaks interactions between ATPs and E, so ΔGATP is +. ΔGIS is - ; sum ΔGATP + ΔGIS favors T form less than in 1 above, so more (or most?) of E is in R form

• T + CP + asp + ATP (cp, asp) – R- ATP (Binding of ATP along with substrates) will have a ΔGATP that is – to go along with the – value for ΔGSB, and this will make the overall ΔG more negative and the affinity higher than in 2 above.

• 5. Inhibition by CTP (analagous to CO2 effect on Hb) keeps more E in T form, makes T---> R harder and makes R---> T more favorable; CTP only binds strongly to T. In T-CTP ---> R + CTP, these are additional interactions that must be broken in T to R. ΔGCTP + ΔGIS for T---> R is more + so T form is even more favored than in 1, and affinity is even lower than in 2.

• 6. Effect of the inhibitor “PALA”, a structural analog of (CP-asp). (Binds at active site, competitive inhibitor)

• a. Binds tightly to R form, less tightly to T form• b. At “low [PALA]” about one molecule of PALA binds

to each E molecule. PALA binding “Pays the price” (ΔGIS) for T---> R: ΔGPALA is – for T + PALA-- R-PALA and ΔG(= ΔGIS + ΔGPALA) is more – than in 1.

• c. At “low [PALA]” and “low [asp] and low [CP]” the rate is greater than in the absence of the inhibitor, because E is in the R form.

Chymotrypsin• Reason to study chymotrypsin: as an example of the

mechanisms and strategies enzymes employ to achieve catalysis.

• Chymotrypsin catalyzes hydrolysis of specific peptide bonds: those preeceeded by large hydrophobic R groups on the substrate. (This is the R group of the residue containing the carbonyl group of the peptide bond that is hydrolyzed)

• How does E achieve this substrate specificity? Hydrophobic/nonpolar/HC side chainsside chains on the E “line” a pocket across from the active site that the hydrophobic side chain on the substrate fits into and interacts with.

• Effects of these interactions:• 1. puts the susceptible peptide bond in extended (β)

conformation so that • 2. the peptide N is positioned close to H of ser OH on E and

the peptide C is positioned close to O of ser OH on E. These are “proximity and orientation effects”.

Catalytic mechanism of chymotrypsin• Step 1:• his N accepts H+ from ser OH, this activates ser O to

attack peptide C, producing the first transient tetrahedral intermediate (TS1)

• Step 2:• his N donates H+ to peptide N, activates breaking of

peptide bond; producing the stable acyl – enzyme intermediate and, product 1 is released

• Step 3:• his N accepts H+ from H2O, activates attack of water

O on peptide C, producing the second transient tetrahedral intermediate (TS2)

• Step 4• his N donates H+ to ser O, activates breaking of E – S

bond, releases 2nd product, returns E to original form

Catalytic mechanism of chymotrypsin• Note that in each of the transition states, the geometry

of the peptide C is tetrahedral and the negatively charge O atom bound to it is positioned in the “oxyanion hole” where it can H bond to two N-H groups of E.

• The peptide C is trigonal planar at all other times and this O isn’t bonded to E. This is another example of specific binding of the TS.

• Catalytic strategies: • 1. proximity and orientation effects • 2. acid-base catalysis• 3. covalent catalysis (E forms covalent intermediate

with S)• Methods of achieving the catalytic effect (lowering

activation energy to speed up the reaction):• 1. specific binding of TS• 2. the mechanism of the reaction catalyzed by the E

has a different mechanism, involving different steps than the reaction without E. Each of these steps has a much lower activation energy than this reaction has without enzyme.

Catalytic mechanism of chymotrypsin

Chymotrypsin employs many of the catalytic strategies that are available to enzymes: 1. proximity and orientation effects 2. acid-base catalysis3. covalent catalysis (forms covalent intermediate with S)But these are NOT the answers to the quiz:

• Of the 2 methods by which it achieves the catalytic effect (increase in reaction rate), one is the same as that of tyrosyl-amino acyl tRNA synthetase. Describe the two methods and give the details for chymotrypsin.

• Compare this slide to slide 28.

• Serine proteases: a group of related enzymes that use the same catalytic mechanism as chymotrypsin

• Trypsin catalyses the same reaction on peptide bonds preceeded by lys and arg side chains: long + charged groups. It has an asp side chain at the bottom of its specificity pocket that can form an ionic bond with the substrate side chain.

Zymogen activation• Chymotrypsin is synthesized as an inactive “proenzyme” (to

avoid digestion of cellular proteins). Specific peptide bonds in it must be hydrolyzed to activate it; (catalyzed by other E’s in the digestive tract).

Reaction coupling• 1. If 2 rxns have a substance in common, they are

coupled: • A + X B ∆G = +2.72 kJ• Z + X Y ∆G = -8.42 kJ• 2. “X” is the common intermediate by which the two

reactions are coupled.• 3. To obtain the coupled reaction, one of the reactions

may need to be reversed so that “X” cancels. • In order to write the coupled reaction that is

spontaneous left to right, one or both reactions must be reversed so that “X” cancels and the sum for ∆G is negative:

• Z + X Y ∆G = -8.42 kJ• B A + X ∆G = -2.72 kJ• Z + B A + Y ∆G = -11.14 kJ

Overview of Metabolism• ATP is consumed in energy requiring

processes and is produced in energy yielding processes.

• G + Pi G6P ΔGo’ = +13.8 kJ • ATP ADP + Pi ΔGo’ = -30.5 kJ • G + ATP G6P + ADP ΔGo’= -16.7 kJ

• PEP Pyr + Pi ΔGo’ = -61.9 kJ• ADP + Pi ATP ΔGo’ = +30.5 kJ • PEP + ADP Pyr + ATP ΔGo’ = -34.4 kJ

Overview of Metabolism• The energy required for the variety of work that an

organism must do (such as muscle contraction, active transport and protein synthesis) is supplied by ATP hydrolysis (H2O omitted in above rxns).

• The amount of ATP in a cell is small and it is constantly consumed, so it must be constantly produced (otherwise, cells like heart muscle and brain/nerves die in a short time).

• The bulk of the energy for ATP production comes from “burning fuel”, the oxidation of food components (fats, carbohydrates (CH2O’s) and proteins). In this oxidation, electrons are transferred from Cs of foods to O2, so we must breathe O2 constantlyconstantly for oxidation and store fuel between meals to maintain fuel supplies.

Overview of Metabolism• The vast majority of ATP in O2 using cells is produced in

oxidative phosphorylation (OP) in a reaction catalyzed by ATP synthase (an enzyme of the inner mitochondrial membrane): ADP + Pi ATP.

• The indirect source of energy for this reaction is a sequence of redox reactions (in the electron transport chain (ET)) that result in O2 reduction: 4H+ + 4e- + O2 2H2O.

• ET produces an “energized” intermediate, which is an elevated [H+] outside the inner mitochondrial membrane that ATP synthase uses as the energy source for OP (Fig 22-29, p821).

Overview of Metabolism• The source of the e-’s in ET is the

C atoms in food molecules. • The e- are transferred from

metabolites of food to NAD+ (made from dietary niacin) and FAD (from riboflavin), which “carry” the e- to ET.

• Some e- transfer to NAD+ and FAD occurs in most metabolic pathways/cycles we’ll study, especially β- oxidation of fatty acids (FAs), but the majority occurs in the TCA cycle (ttriccarboxylic aacid).

• The main purpose of the TCA is to oxidize C: “harvest” the e- of food fuels for delivery to ET. (Fig 22-1 p 798)

Overview of Metabolism

• So the overall scheme for ATP production is to convert food molecules to the form in which they are consumed by the TCA cycle, acetyl coenzyme A (ACoA) (Fig 16-3 p 551).

• CH2O’s are polymers which are hydrolyzed to glucose (G) (+/or other sugars which are converted to G). G is converted in glycolysis to pyruvate, which is converted to ACoA in the pyruvate dehydrogenase (PDH) rxn.

• Fats (triglycerides or triacylglycerols) are hydrolyzed to fatty acids (FAs) which are converted in β-oxidation to ACoA.

• Proteins are hydrolyzed to AAs which are converted in AA oxidation to ACoA directly or via other metabolites like pyruvate.

• So the metabolism of the major food groups involves separate pathways which converge in production of ACoA, the fuel of the TCA cycle.

Overview of Metabolism• Aside from ATP production, our other main concern will be

storage of fuel after a meal and its release as needed. • AAs are stored as muscle protein and are released in short

term (> 8 hr) CH2O starvation. • FAs are stored as fat (which is the major stored fuel by far) and

released continuously since they are the main fuel for they are the main fuel for most cells most of the timemost cells most of the time.

• G is stored as the polymer glycogen and released in muscle cells to do muscle work and in liver for export to the blood to supply the brain its preferred fuel.

• G is also produced from AAs in gluconeogenesis for export to blood in times of CH2O starvation.

• Since the capacity to synthesize and store glycogen is limited, much of the G in a high CH2O meal is converted to fatty acids via glycolysis, PDH and FA synthesis.

Overview of Metabolism• The pathways and cycles mustmust work together. • For brain to function the nerve cells must get ATP

from the sequential action of glycolysis, then PDH, then TCA, then ET and OP.

• Glycogen breakdown and gluconeogenesis work in concert to increase blood G starting a few hours after a meal.

• Glycolysis does not convert G pyr at a high rate when gluconeogenesis is converting pyr G at a high rate.

• Also, a given pathway may have many functions: glycolysis is not only involved in ATP production and feeding FA synthesis, it also feeds AA synthesis and other biosynthetic pathways and the production of TCA intermediates (as distinct from ACoA).

Overview of Metabolism• So, how does a pathway “know” when to “stop” and

when to “go”? • We will focus on 2 mechanisms: • allosteric regulation of specific enzymes by effectors

that indicate the need (or lack thereof) in that cell for their activity;

• and control of activity by reversible phosphorylation in response to the hormones insulin, adrenaline and insulin, adrenaline and glucagon. glucagon.

• These hormones signal the cells, especially liver cells as to the needs of the organism.

• CO2 regulation of Hb is a molecular model for both of these mechanisms.