BIOC 460, Spring 2008LECTURES 13-14, Enzymes - CatalyticStrategies 1Lectures 13-14Enzymes: Catalytic StrategiesReading: Berg, Tymoczko & Stryer, 6th ed.,Chapter 9, pp. 241-254hexokinase conformational change (Jmol):http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/hexokinase/newhk.htmlmovie of chemical mechanism of serine proteases (from Voet & Voet,Biochemistry, 3rd ed., 2004, Wiley):http://www.biochem.arizona.edu/classes/bioc460/spring/460web/lectures/LEC13-14_EnzCatMech/15-3c_SerineProtease-b3/SerineProtease.htmSerine proteases (Jmol)http://www.biochem.arizona.edu/classes/bioc462/462a/jmol/serprot/serprot1.htmKey Concepts• Mechanisms used by enzymes to enhance reaction rates include:(1st 4 mechanisms based on BINDING of substrate and/or transition state)1. Proximity & orientation2. Desolvation (one type of electrostatic catalysis)3. Preferential binding of the transition state4. Induced fit5. General acid/base catalysis6. Covalent (nucleophilic) catalysis7. Metal ion catalysis8. (Electrostatic catalysis)• The chemical mechanism of serine proteases like chymotrypsin illustrates:– Proximity and orientation– Transition state stabilization– Covalent catalysis, involving a “catalytic triad” of Asp, His and Ser inthe active site– general acid-base catalysis– electrostatic catalysisLearning Objectives• Discuss (briefly explain): 8 general catalytic mechanisms used by enzymesto increase the rates of chemical reactions. (You won't be asked on anexam to simply LIST them, but you could be expected to explain any one --or 2 or 3 or 4 -- of them.)• Terminology: proteolysis, serine protease, general acid, general base,catalytic triad, acyl group, tetrahedral intermediate, acyl-enzymeintermediate, acylation, deacylation, nucleophile, oxyanion hole• Explain why peptide bonds are kinetically stable in the absence of a catalyst,given that equilibrium lies far in the direction of hydrolysis in 55.5 M H2O.(Why is any specific reaction a slow reaction?)• Describe the chemical mechanism of hydrolysis of peptide bonds bychymotrypsin, including the following:– What is the "job" of the catalyst (the protease), i.e., what group needs tobe made more susceptible to nucleophilic attack?– Describe substrate binding, including the role and chemical nature of the"specificity pocket" in chymotrypsin, and which peptide bond in thesubstrate (relative to the specificity group) will be cleaved.– Draw the structure of the catalytic triad at the beginning of the reaction,and explain how the states of ionization and hydrogen bonding pattern ofthose 3 groups change step by step during catalysis.Learning Objectives, continued• (chemical mechanism of chymotrypsin, continued)– Explain the role of each member of the catalytic triad in the reaction.– Identify the nucleophile that attacks the carbonyl carbon in acylation;identify the nucleophile that attacks the carbonyl carbon indeacylation.– Describe the acyl-enzyme intermediate, including identifying thetype of bond attaching the acyl group to the enzyme (Is it an amidelinkage? anhydride? ester? etc.) and how that acyl group relates tothe structure of the original substrate.– Draw the structures of each of the tetrahedral intermediates in thereaction. (If you can do this, you understand the chemistry by whichthey formed.)– Identify the leaving group coming from each of the tetrahedralintermediates as the intermediate breaks down.– State what is being acylated and deacylated in the chymotrypsinreaction (be specific about the functional group involved).– Explain the role of the "oxyanion hole" in the mechanism.– Describe which type(s) of general catalytic mechanisms (firstlearning objective above) are used by chymotrypsin, and how.Learning Objectives, continued• Compare (very briefly, just the “bottom line”) the overall 3-dimensionalstructures of chymotrypsin, trypsin, and elastase, and compare thesubstrate binding specificities of those 3 enzymes, explaining therelationship of the “specificity site/pocket” structure to the differences insubstrate specificity..• How do 3 other classes of proteases (besides the serine proteases)generate nucleophiles potent enough to attack a peptide carbonyl group?• To which protease class does HIV protease belong? Describe thequaternary structure and symmetry of the HIV protease and where in thequaternary structure the active site residues are located.General Catalytic Mechanisms• Different enzymes use different combinations of mechanisms to reduceactivation energy and thus increase rate of reaction.• 7 (or 8) "types" of mechanisms below -- really "overlapping" concepts inmany cases.• 1st 4 mechanisms related to BINDING of substrate and/or transition state,(reaction takes place in active site, not in bulk solution)1. PROXIMITY AND ORIENTATION (catalysis by “approximation”)– Proximity• Reaction between bound molecules doesn't require an improbablecollision of 2 molecules.• They're already in "contact" (increases local concentration ofreactants).– Orientation• Reactants are not only near each other on enzyme, they're orientedin optimal position to react.• The improbability of colliding in correct orientation is taken care of.BIOC 460, Spring 2008LECTURES 13-14, Enzymes - CatalyticStrategies 2General Catalytic Mechanisms, continued2. DESOLVATION• Active site gets reactants out of H2O.• Lower dielectric constant environment than H2O (more nonpolarenvironment), so stronger electrostatic interactions (strength inverselyrelated to dielectric constant).• Reactive groups of reactants are protected from H2O, so H2O doesn'tcompete with reactants.– H2O won't react to give unwanted byproducts, e.g., by hydrolysis of somereactive intermediate in the reaction that was supposed to transfer itsreactive group to another substrate.3. TIGHT TRANSITION STATE BINDING• used to be called "strain and distortion"• Enzyme binds transition state very tightly, tighter than substrate.• Free energy of transition state (peak of free energy barrier on reactiondiagram) is lowered because its "distortion" (electrostatic or structural) is"paid for" by tighter binding of transition state than of substrate.4. INDUCED FIT• Conformational change resulting from substrate binding• Binding may stabilize different conformation of enzyme or substrate or both.• Conformational change– orients catalytic groups on enzyme,– promotes tighter transition state
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