UVM CHEM 205 - Mechanisms of Enzyme Action

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• Stabilization of the Transition State• Enormous Rate Accelerations• Binding Energy of ES• Entropy Loss and Destabilization of ES• Types of Catalysis• Serine Proteases• Aspartic Proteases• LysozymeMechanisms of Enzyme ActionStabilizing the Transition State• Rate acceleration by an enzyme means that the energy barrier betweenES and EX‡ must be smaller than the barrier between S and X‡• This means that the enzyme must stabilize the EX‡ transition state morethan it stabilizes ESEA cyclohexane “flippase” would bind more tightly to the eclipsed part of thetransition state than to either of the staggered ground state conformers1Rate Acceleration in Enzyme-Catalyzed Reactions• Mechanisms of catalysis:•– Entropy loss in ES formation– Destabilization of ES– Covalent catalysis– General acid/base catalysis– Metal ion catalysis– Proximity and orientation2Binding Energy of ESCompeting effects determine the position of ES on the energy scale• Try to mentally decompose the binding effects at the active site into favorableand unfavorable• The binding of S to E must be favorable• But not too favorable!• Km cannot be "too tight" - goal is to make the energy barrier between ES andEX‡ small3Entropy Loss and Destabilization of ESraising the energy of ES raises the rate• For a given energy of EX‡, raising the energy of ES will increase the catalyzed rate• This is accomplished by•– a) loss of entropy due to formation of ES– b) destabilization of ES by• strain• distortion• desolvation4Covalent Catalysis• Enzyme and substrate become linked in a covalent bond at one or morepoints in the reaction pathway• The formation of the covalent bond provides chemistry that speeds thereaction1. ChymotrypsinElastaseEsterasesSubtilisinThrombinTrypsin2. G-3-P dehydrogenasePapain3. Alkaline phosphatasePhosphoglucomutase4. Phosphoglycerate mutaseSuccinyl-CoA synthetase5. AldolaseDecarboxylasesPyridoxal phosphate-dependentenzymesHNOORHNOHOOHNSORHNSHOOHNO PO32-HNOHOOHNONNHHNONNPO32-RNH3+RNH+acyl-seracyl-cysphosphoserinephosphohistidineSchiff base5The Enzyme as NucleophileG-3-P dehydrogenaseHNSORHNSHOOacyl-cys6General Acid-base Catalysisa proton is transferred in the transition state• "Specific" acid-base catalysis involves H+ or OH- that diffuses into the catalyticcenter• "General" acid-base catalysis involves acids and bases other than H+ and OH-• These other acids and bases facilitate transfer of H+ in the transition stateSpecific acid-base catalysis General acid-base catalysisAn ionizable group on a protein will be most effective as a H+ transferagent at or near its pKaBiochemistry usually happens near pH7, where histidine is the mosteffective general acid or base (imidazole pKa = 6)7Low-Barrier Hydrogen Bonds• Typical O - O distance in C=O…..H-O is 2.8 Å• O-H is 1 Å, H-bond is 1.8 Å• Bond order ~0.07• Typical bond strength 10-30 kJ/mol• Protein structure can constrain H-bond donor and acceptor to be close• O - O distance may be as low as 2.3 Å• When there is no barrier to H exchange, the interaction is a low-barrier H-bond• Typical LBHB strength may be 60 kJ/molEHHO OO OO OO OH H2.8 Å 2.5 Å 2.3 Å• LBHBs require matched donor/acceptor pKas• A weak H-bond in E or ES may become a LBHB in an E’S intermediate or inEX‡8The Serine ProteasesTrypsin, chymotrypsin, elastase, thrombin, subtilisin, plasmin...• All involve a serine in catalysis - thus the name• Ser is part of a catalytic triad of ser, his, asp• Serine proteases are homologous, but locations of the three crucial residues differsomewhat• Enzymologists agree, however, to number them always as his57, asp102, ser1959SubstrateSpecificity in the Serine Proteases10Experimental Evidence for Mechanism• Most studies use artificial substrates• p-nitrophenylacetate cleaved to p-nitrophenolate (lmax = 400 nm)• At high [E], a rapid burst of p-nitrophenolate is observed• Followed by slower, steady-state hydrolysis11Burst-phase kinetics• Evidence for a 2-step mechanism• Fast first step• Slower second step• E + A E’P + H2O Q + E• Fast when [E’P] is v. small• Slows down until E is saturated by E’Prds12Serine Protease MechanismA mixture of covalent and general acid-base catalysis• Asp102 functions only to orient his57• His57 acts as a general acid and base• Ser195 forms a covalent bond with peptide to be cleaved• Covalent bond formation turns sp2 C into sp3• The tetrahedral oxyanion intermediate is stabilized by NH of gly193 and ser19513A Detailed Mechanism for Chymotrypsin1415The Aspartic Proteases pepsin, chymosin, cathepsin D, renin and HIV-1 protease• All involve two asp residues at the active site• Two asps work together as general acid-base catalysts• Most aspartic proteases have a tertiary structure consisting of two lobes(N-terminal and C-terminal) with approximate two-fold symmetry• HIV-1 protease is a homodimerHIV ProteasePepsin16Aspartic Protease Mechanismthe pKa values of the asp residues are crucial• One asp has a relatively low pKa, other has a relatively high pKa• Deprotonated asp acts as general base, accepting a proton from H2O, formingOH- in the transition state• Other asp (general acid) donates a proton, facilitating formation of tetrahedralintermediate• What evidence exists to support thehypothesis of different pKa values forthe two asp residues?• If activity increases with increasingpH, there is likely a general base atthe active site–can’t function when protonated(low pH)• If activity decreases with increasingpH, there is likely a general acid atthe active site–can’t function whendeprotonated (high pH)• If both, we get a bell-shaped activityprofile•Curve fitting allows an estimate of pKas•In pepsin, one asp has pKa of 1.4, the other 4.3•This simple model was modified in 2000…17A Mechanism for Asp ProteasesHOHOOCOOCHHOHOOCOOCHRNROHSHOHOOCOOCHRNRO-HHOHOOCOOCHRNRO-HHOHOOCOOCHRNROHHO-HOOCOOCHRNROHH HOOCOOCHNRHRNH3+H2O18HIV-1 Proteasea novel aspartic protease• HIV-1 protease cleaves the polyproteinproducts of the HIV genome• This is a remarkable imitation ofmammalian aspartic proteases• HIV-1 protease is a homodimer - moregenetically economical for the virus• Active site is two-fold symmetric• Mechanism doesn’t need different pKasWhat does HIV Protease do?19Therapy for HIV?protease inhibitors as AIDS drugs• If the HIV protease can be


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UVM CHEM 205 - Mechanisms of Enzyme Action

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