Lecture 15: Enzymes & Kinetics MechanismsMargaret A. DaughertyFall 2004ROLE OF THE TRANSITION STATEConsider the reaction:H-O-H + Cl- H-Oδ-•••H•••Clδ- HO- + H-ClReactantsTransition stateProductsIn the transition state (denoted by X‡), the chemistry is intermediatebetween reactant and product. The substrates are strained… hencechemistry can occur.Enzymes work to reduce ∆G‡, the barrier between substrate and transitionstate.REGULATION AND RECOGNITIONBIOC 205LOCK & KEYLysozyme binding toan antibodyINDUCED FIT(enzymes)Hexokinasebinding it’ssubstrateglucoseENZYME-SUBSTRATE INTERACTIONSTHE LOCK & KEY MODELA perfect match between enzyme and substratecan explain enzyme specificity does not explain enzymatic catalysisImplies a rigid, inflexible enzymeIf everything is such a perfect fit, how does the chemistry occur? How do we go from substrate to product?ENZYME-SUBSTRATE INTERACTIONSTHE INDUCED FIT MODELKEY FEATURES:ENZYME STRUCTURE CHANGES IN PRESENCE OF SUBSTRATE --- BRINGS CATALYTIC GROUPS INTO CORRECT POSITION TO DO CHEMISTRY ---SUBSTRATE IS FORCED INTO TRANSITION STATE CONFORMATIONACCOUNTS FOR SPECIFITY AND FOR CATALYSISIntermediate State in Catalysisuncatalyzed reactionCatalyzed reaction: 2 low barriers replacing 1high barrier.Lower activation energy; reaction goes fasterES complex with low energyEX≠ complex with lower energy than X≠10-13 secEnzymes stabilize transition state more than ES complexLARGE 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 accelerationActive Site• Small compared to the protein;• 3D entity, usually found in a cleft orcrevice;• Size, shape, charge, non-covalentinteractions are all important!• Binding is via multiple interactions;• Specificity achieved by the arrangement ofthe active site;• Converts substrate to product.ActivesiteENZYME ACTIVE SITES BIND THE TRANSITION STATE BETTER THANSUBSTRATE OR PRODUCTGoal: Perform chemistry! Fast! Binding of Substrate must be favorable;……...but not too favorable!Destabilization of ES relative to the transition state, EX‡Loss of entropy on formation of ESDestabilization of ES by StrainDistortion DesolvationFig 14.2Favorablebinding energydestabilizedby entropyloss, strain,distortion,desolvation!Note: orientation:the active site hasthe catalytic groupsin the most effectiveorientation to carryout the neededchemistry.FORMATION OF ES: LOSS OF ENTROPY MAIN POINTS:2 reactants --> 1 productLose translational and rotational entropyEntropy offset by favorable ∆H (chemical interactions)No further loss of entropy in going to EX‡DESTABILIZATION OF ES: Strain, Desolvation, Electrostatic EffectsMAIN POINTS1). Active site is specialized to bind transition state to carry out chemistry;in order to make a “fit” the ES complex is strained or destabilized.2). When charged groups move from solvent to active site, they often becomedesolvated - this makes them less stable, hence more reactive.+Note: proximityBringing the substrate together with the catalytic groups on the enzyme results inan effective concentration increase relative to the concentration of substrates insolution. Hence faster reaction rates.MAIN POINTCharged groups on S may be forced to interact with “like” charges. Thisis unfavorable and destabilizes S. Reaction pathway acts to remove stress- hence faster rates result.DESTABILIZATION OF ES: Strain, Desolvation, Electrostatic EffectsSTUDYING TRANSITION STATE - TRANSITION STATE ANALOGS:High affinity (10-14 M) substrates that mimic the transition state; transitionstate analogs are stable and only mimic the actual TS. As such they neverbind as tightly as the TS itself!AAs Frequently Involved inCatalysisCATALYTIC FUNCTIONS OF REACTIVEGROUPS OF IONIZABLE AMINO ACIDSR-C- Acyl groupOCOVALENT CATALYSISA covalent bond is formed between enzyme and substrateBX + Enzyme E-B + X + Y Enz + BYserine proteases: coming right up!Acid-Base CatalysisSpecific vs. generalSpecific Acid-Base Catalysis: H+ or OH- accelerates the reaction;donated from H20In this case, the reaction will be pH dependent;However 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 are characterized bychanges in rate withincreasing bufferconcentrations - i.e., there issome other group available toact as an general acid orgeneral base.Histidine plays a major role as a general acid or base. pKa ~ 7.0Enolase: Lys - general base;abstracts H+Glu - general acid;Donating proton to OH --> H20Metal 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!Im = imidazole of histidineTHE 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: