Lecture 12: Enzyme CatalysisSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Lecture 12:Enzyme CatalysisCatalytic StrategiesSteps in a ReactionEnzymes are Classified According to the Reactions They CatalyseBut while the details of the particular reaction vary fromenzyme to enzyme, similar strategies are used to carry them out.Common Types of CatalysisCovalent Catalysis: A group on the enzyme becomes covalently modified duringreaction, e.g. by forming a covalent bond to the substrateduring the reaction.General Acid-Base Catalysis:A group on the enzyme acts as an acid or base: it removes aproton from or donates a proton to the substrate during the reaction.Metal Ion Catalysis:A metal ion is used by the enzyme to facilitate a chemicalrearrangement or binding step.Catalysis by Approximation:The enzyme holds two substrates near in space and in preciselythe correct spatial orientation to optimize their reaction.(Most enzymes use a combination of several of these strategies)Enzyme SubstrateCovalentIntermediateEnzyme ProductsCovalent CatalysisRate acceleration by transient formation of a COVALENT enzyme-substrate bondReaction can’t go back because displaced group has been released.The enzyme alters pathway to get to product by stabilizing the intermediate with a covalent bond. But: if a covalent bond is formed at an intermediate step, the bond must be broken in a subsequent step to finish the reaction.Nucleophile: an electron-rich group that attacks nuclei.Examples of nucleophiles among protein functional groups:unprotonated His imidazoleunprotonated -amino groupunprotonated sidechain amino group of Lysthiolate anion (-S-) of Cysaliphatic -OH of Sersidechain carboxylates of Glu, Asp(Also some coenzymes.)Covalent Catalysis Requires a Highly Reactive GroupSome chemical group that can invade the substrate- usually a nucleophile.General Acid-Base CatalysisSpecific functional groups in enzyme structure are positioned to eitherDonate a proton (act as a general acid)or Accept a proton (act as a general base).This enables enzyme to avoid unstable charged intermediates in reaction,so as to keep the transition state in a stable (low-energy) state But: A group that donates a proton (acts as a general acid) in catalysis has tothen accept a proton (act as a general base) later in catalytic mechanismfor catalyst to be regenerated in original form.Examples of general acid/base catalysts among protein functional groups:His imidazole-amino groupthiol of CysR group carboxyls of Glu, AspSidechain amino group of LysAromatic OH of TyrGuanidino group of ArgMetal Ion CatalysisMetal ions can be used in a variety of ways by enzymes. (In fact they are souseful that about one-third of enzymes use them for one thing or another.) Binding and orientation of substrate:By forming strong ionic interactions with substrate, it can be precisely oriented. (Especially strong because when water is excluded from active site, the dielectric constant is quite low.)Redox reactions:Ions that have more than one possible chargestate (eg. iron Fe2+ and Fe3+) can gain or lose electrons duringthe reaction, avoiding unstable charged intermediates.Shielding or stabilizing negative charges: If charge on substrate or on transition state is an integralpart of the reaction, the enzyme can form strong ionic interactionswith that charge which are stabilizing.Enzyme SubstrateUnstableStable+2--+2++30Binding and orientation of substrateAND/ORShielding or stabilizing negative chargesRedoxReactionsCatalysis by ApproximationProximity: Reaction between bound molecules doesn't require an improbablecollision of 2 molecules. They're already in "contact" (increases localconcentration of reactants).Orientation: Reactants are not only near each other on enzyme, they'reoriented in optimal position to react. The improbability of collidingin correct orientation is taken care of.Substrates held close in spaceand in correct orientationResults in moreefficient reaction pathwayOther Effects that Stabilize the Transition StateElectrostatic Effects:Increase in strength of ionic interactions due to lowerdielectric constant.Desolvation:Exclusion of water from active site.Induced Fit:Change in conformation of enzyme or substrate tooptimize interactions.Providing a lower dielectric constant of the environment in theactive site (hydrophobic environment)Altering pK values of specific functional groups.Stabilizing a particular conformation of the critical groups inthe active site by electrostatic interactions.Stabilizing (binding) a charged intermediate or transition state byproviding an oppositely charged enzyme group close by.Electrostatic EffectsEnhancement of the attraction between opposite chargesby various means. Examples:221DrqqkF Coulomb’s Law:Lower dielectric constant environment than in water.results in stronger electrostatic interactions.Reactive groups of reactants are protected from H2O,so H2O doesn't compete with reactants or affect equilibrium.DesolvationBy sequestering reactants away from water, two major effects areAchieved:Conformational change can: Promote faster chemical steps(eg by orienting catalytic groups on enzymePromote tighter transition state binding(eg by orienting binding groups on enzyme)Close off active site and sequester reactants away from water.Induced FitConformational change resulting from substrate binding may stabilize a different conformation of either enzyme or substrate or both.Examples:Chymotrypsin- hydrolysis of peptide backboneIncorporates covalent catalysis and acid-base catalysis(details next lecture)Carbonic Anhydrase- equilibrate carbon dioxide and carbonic acidMetal ion catalysis(near catalytic perfection)Restriction Enzymes- sequence-specific cleavage of DNA backbonehydrolysis via covalent catalysis(extreme specificity)NMP Kinases- transfer of phosphate group between 2 substratesgroup transfer using metal ion catalysis(very efficient- avoid loss of the phosphate group)Reactions Proceed in a Series of StepsS PNet Reaction:SubstrateBindingChemicalRearrangementProductReleaseE + S ES EP E + PEach of these steps might be analysed further to understand atomic details.Enzyme Catalysed Pathway:A + B P + Q Group Transfer Reactions Have 2 SubstratesS1-G + S2 S1’ + S2’-G 2 Substrates 2 ProductsSubstrates and Products can be bound and released in various
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