Factors that Affect Enzyme Catalysis

1 1 Factors that Affect Enzyme CatalysisIonic strength: = (Zi2ci)Review: urea, guanidiniumhydrochloride, detergents, or organic solventsTemperaturepHFactors that Affect Enzyme Catalysis : Ionic strength: = (Zi2ci)NaCl (mM) (107M-1s-1) with O2-for active Cu2+Cu+2+ O2- Cu++ O2Cu++ O2-+ 2 H+ Cu+2+ H2O2 Superoxide Dismutase2 Conditions that cause protein denaturation: of urea orguanidiniumhydrochloride (G@@HCl)-Urea and G@@HClboth H-bond to proteins causingdisruption of the H-bond unfold and remain soluble (usually). denaturation renaturation Folded proteinC O 2-N H 3+RRRU nfolded proteinC O 2-RRRN H 3+COH2 NNH2 CNH2+H2 NNH2Cl-Conditions that cause protein denaturation: Soaps/Detergents denaturation renaturation Folded proteinC O 2-N H 3+RRRU nfolded proteinC O 2-RRRN H 3+Addition of soaps or detergentsNonpolarportions of soaps and detergents interact with protein R groups causing the loss of the hydrophobic denature and remain structures formed insodium dodecylsulfate (SDS)C O 2-RRRN H 3+3 Conditions that cause protein denaturation.

2 Organic Solvents denaturationprotein precipitateswith poorly definedstructure renaturation see belowC O 2-RRRN H 3+C O 2-RRN H 3+RFolded proteinC O 2-N H 3+RRRU nfolded proteinC O 2-RRRN H 3+Addition of water-soluble organic solventsSolvent molecules interact with protein R groups causing the loss of the hydrophobic denature and often that cause protein denaturation: H+/OH-Extremes of pH-OH-and H3O+both H-bond to proteins causing disruption of the H-bond proteins may unfold and aggregate into denatured precipitates (solids). This is more common at low pHs. denaturationprotein precipitateswith poorly definedstructure renaturation see belowC O 2-RRRN H 3+C O 2-RRN H 3+RFolded proteinC O 2-N H 3+RRRU nfolded proteinC O 2-RRRN H 3+HOHHHO4 Factors that Affect Enzyme Catalysis : that Affect Enzyme Catalysis : pH(NOT EXTREMES OF pH)Fig 8-17a,bBell-shaped pH-rate profilesThe Enzyme active site has a minimum of two functional the maximum rate:One group is required in the conjugate acid form,AHOne group is required in the conjugate base state, B:rate = k[E-AH][E-B:]5rate = k[E-AH][E-B:]Fig 8-17a,b+H:B-E-AH :B-E-AH :B-E-A-inactiveactiveinactiveExamples of B:= -HIS.

3 , -CO2-, -S-, -NH2, etcExamples of AH= -HISH+, -CO2H, -SH, -NH3+, etcActive form has a maximum concentration at the top of the bell-shaped profileWhat amino acids are likely to be involved in Catalysis ?Fig 8-17a,b+H:B-E-AH :B-E-AH :B-E-A-inactiveactiveinactiveThe pKavalues for the functional groups involved in Catalysis can be estimated by looking at the pH values on either side of the bell profile at maximum velocity6 PEPSIN?Fig 8-17+H:B-E-AH :B-E-AH :B-E-A-inactiveactiveinactivepKavalues indicate very acidic groups-ASP --CO2H-GLU --CO2 HGLUCOSE-6-PHOSPHATASE?Fig 8-17+H:B-E-AH :B-E-AH :B-E-A-inactiveactiveinactivepKavalues indicate what amino acids?pKa~ 6 HIS (what form?)CB since rate 88with pH 88pKa~ or N-terminal (what form?)CA since rate 99with pH 887 ChymotrypsinRate%%?

4 ChymotrypsinRate%%E-B:What amino acid side chain and what form of this side chain (CA or CB) accounts for this increase in rate as pH is raised?+ H+pKa = HCH2 HNNrate %%[E-HIS:]E-HIS:H+inactiveE-HIS:active8 ChymotrypsinWhy does rate level off at high pH?+ H+pKa = HCH2 HNNrate %%[E-HIS:]E-HIS:H+inactiveE-HIS:active[E o] <<<< [So] at all So; Eo is the limiting all of the E-HISis in the CB form, the maximum amount of the CB form is the rate [E-HIS], the rate reaches a maximum BASIS OF Enzyme Catalysis In the attached schemes, the Enzyme active site is schematically represented with a bold-lined "box".The amino acid side chains that act as functional groups i the site are written in bold lines and lettersand are attached to the "box". Substrates are indicated in plain Enzyme "box"for chymotrypsin:ORCOOCH2 OHNNHR'NHCE xample substrate:NOTATION: hydrogen bonding or other weak interactions mechanistic arrowsProblem Set: Notation9 Problem Set: Catalytic Triad Catalytic Triad of Serine HIS.

5 SERP roblem Set: Sample Step #1 CHYMOTRYPSINORCOOCH2 OHNNHR'NHCORCOOCH2 OHR'NHCHNNHOASPCBHISCBSERNuPeptide SubstrateCovalentIntermediate10 Problem Set: Sample Step #2 ORCOOCH2 OHR'NHCHNNH2OR'NH2 HNNORCOOCH2 OCHOHASPCBHISCASERC ovalentIntermediateProblem Set: What is function of ASP?CHYMOTRYPSINORCOOCH2 OHNNHR'NHCORCOOCH2 OHR' HIS HIS:H+pKamaking CA-HIS:H+a poorer acid, but the CB-HIS: a better base to attach the very weak acid HO-SER!11 Problem Set: Predict pH-Rate ProfileCHYMOTRYPSINORCOOCH2 OHNNHR'NHCORCOOCH2 OHR'NHCHNNHOD eacylationof Chymotrypsin: pH-Rate Profile2 HNNORCOOCH2 OCHOHORCOOCH2 OHHOCHNN12EE'ES2P2S1P1 CHYMOTRYPSIN Catalysis OFPEPTIDE AND ESTER HYDROLYSISPING PONG BI BI MECHANISMMore about ChymotrypsinPing Pong Mechanism!fastslowacyl- Enzyme intermediateS1NR'HHE'OCORRCOOHS2P1P2H2 OEOHEOHserRHNR'OCFast = acylation stepSlow = deacylationstepAcyl Enzyme = inactive13[P1] t = o = [Eo] = active sites[P1]time (min) formation of P1slow formation of P1ES1P1fastE'S2P2slowUnderstanding Laboratory ExperimentRead Text about ChymotrypsinFig 8-2014 Read Text about RelationshipsBox 8-3B compared to A: B faster, tigherbindingC compared to B: C faster, weaker binding15 Enzyme ActivityVo (rate) at given Eohas units of M min-1 Specific Activity= Rate/mgEoM min-1U--------=----mg mg1 U often defined as 1 mmmol/mi