Lecture 16:Enzymes & Kinetics V: MechanismsMargaret A. DaughertyFall 2003Enzyme function: the transition stateCatalytic ReactionsCatalysts (e.g. enzymes) act by lowering the transitionstate free energy for the reaction being catalyzed.A BIntermediate State in CatalysisUncatalyzedreactionCatalyzedreaction10-13 secLARGE RATE ACCELERATIONS:ENZYME STRUCTURE AND MECHANISMMECHANISMS OF CATALYSIS1). Entropy loss in the formation of ES2). Destabilization of ES due to strain, desolvation or electrostaticeffects3). Proximity and orientation4). Covalent catalysis (an example of the serine proteases)5). Metal ion catalysis6). General acid or base catalysisAny or all of this things can contribute to catalyticrate accelerationNote: orientation ofactive site is optimalfor chemistryFORMATION OF ES: LOSS OF ENTROPY MAIN POINTS:Entropically unfavorableEnthalpically favorableNo unfavorable entropy in ES --> EX‡DESTABILIZATION OF ES: Strain, Desolvation, Electrostatic EffectsMAIN POINTS1). Active site is specialized to bind transition state to carry out chemistry2). When charged groups move from solvent to active site, they often becomedesolvated.+Note: proximityBringing the substrate together with the catalytic groups on the enzyme results inan effective concentration increase relative to the concentration of substrates insolution.MAIN POINTCharged groups on S may be forced to interact with “like” charges. Thisis unfavorable and destabilizes S.DESTABILIZATION OF ES: Strain, Desolvation, Electrostatic EffectsSTUDYING TRANSITION STATE ANALOGS:HIGH AFFINITY (10-14 M) SUBSTRATESTHAT MIMIC THAT THETRANSITION STATEAAs Frequently Involved inCatalysisCATALYTIC FUNCTIONS OF REACTIVEGROUPS OF IONIZABLE AMINO ACIDSR-C- Acyl groupOAcid-Base CatalysisSpecific Acid-Base Catalysis: H+ or OH- accelerates the reaction;donated from H20However buffers that can donate or accept H+/OH- will not affect thereaction rate:Acid-Base CatalysisGeneral Acid-Base Catalysis: in which an acid or base other than H+or OH- (other than H2O) accelerates the reaction; reactive groups inthe enzymes active sites;These arecharacterizedby changes inrate withincreasingbufferconcentrationsGENERAL ACID/BASE CATALYSIS UNCATALYZEDGENERAL BASEGENERAL ACIDEnolase:Metal Ion CatalysisMetalloenzymes: bind metal tightly require metal for 3-D structure Transition metal ionsFe2+, Fe3+, Zn2+, Mn2+ or Co2+Metal activated enzymes: bind metals weakly; usually onlyduring catalysis - play a structural role; bindmetals from solutionalkali and alkaline earth metalsNa+, K+, Mg++ or Ca++Roles: Bind to substrates and orient the substratesMediate redox reactions through reversible changes in themetals oxidation stateElectrostatically shield or stabilize negative charges.Human Carbonic Anhydrase: A Zinc containing enzymeCO2 + H2O <---> HCO3- + H+Zinc is tetrahedrally coordinatedH2O is polarized!THE SERINE PROTEASESTrypsin, chymotrypsin, elastase, thrombin, subtilisin,plasmin, TPAAll involve serine in their catalytic mechanism;Serine is part of a “catalytic triad” of Ser, His, AspAll serine proteases are homologous, but locations of thethree critical residues vary.By convention, numbering of critical residues is always thesame: His-57, Asp-102 and Ser-195PRIMARY STRUCTURE OF SERINE PROTEASESZYMOGENS ARE CLEAVED TO THEIR ACTIVE CONFORMATIONACTIVE SITE: A DEPRESSION ON PROTEIN SURFACEChymotrypsin in complex with eglin C“depth” of active site depression depends on reactionChymotrypsin, trypsin and elastaseblue yellow greenAll three proteases show: similar backbone conformations active site residue orientationsyet…. All three exhibit different cleavage specificityChymotrpysin: aromatics; trypsin: basic & elastase: gly & alanineSUBSTRATE SPECIFICITYshallow hydrophobicdeephydrophobicdeepnegatively chargedTRYPSIN - TRYPSIN INHIBITOR COMPLEXARTIFICIAL SUBSTRATES PROVIDE INSIGHTINTO MECHANISMEVENTS AT THE ACTIVE SITE: MECHANISMCOVALENT&GENERAL ACID-BASE CHEMISTRY1). Asp-102 functions to orient His-572). His-57 acts as a general acid and ageneral base.3). Ser-195 covalently binds thepeptide to be cleaved4). Covalent bond formation turns atrigonal C into a tetrahedral carbon5). A tetrahedral “oxyanion”intermediate is stabilized by NH’s ofGly193 and Ser195.MECHANISM I: BINDING OF SUBSTRATESubstrate C=0 hydrogen bondsto amide Hs of G193 and S195“Oxyanion Hole” Major Role in Transition State StabilizationRecall: binding site is relatively hydrophobic; need to neutralize charges Enhanced stabilization in thetransition stateMECHANISM II: ATTACK OF WATER; RELEASEOF AMINO PRODUCTFrom last slideMECHANISM III: COLLAPSE OF TETRAHEDRALINTERMEDIATEMECHANISM IV: RELEASE OF CARBOXYLPRODUCTReview1). Enzymes stabilize transition state more than ES complex.2). There are a limited number of factors that contribute to catalysis.3). Destabilization of ES relative to the transition state occurs via:Loss of entropy on forming ESStrain on the ES complexDistortion of the ES complexDesolvation of the ES complex4). Catalysis is also achieved through orientation of the chemically reactivegroups on the enzyme in close proximity with the substrate.5). Transition state analogs are a way of studying enzyme mechanism.6). The serine proteases have a catalytic triad that consists of Ser, Hisand Asp.The job of Asp102 is to correctly position His57 in the active site.His57 acts both as a general acid and a general base.Serine covalently binds the substrate.Water is involved in the