BU BI 421 - Lesson 5 Enzyme Catalysis

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Mechanisms of enzyme catalysis Acid base Gives takes protons Reactant species When proton transfer to from H2O Faster than the rate of break down of intermediates presence of other proton donors or acceptors does not increase rate of reaction R3 0 4 0 H C R2 N H 4 11 R2 N H 4 B A BH OH 1201 1 1 HOH HOH ppl 4 f 0 4 0 R2 H NIH R3 ra t 1 1 4 01 0 1 R 1 R R2 i t N R4 Products Without catalysts unstable charged intermediate breaks down rapidly forms reactants When proton transfer toorfromtlzois slower than the rate of break down of intermediates only a fraction of the intermediates formed a a g presence of alternative proton donors HA or acceptors CB increases the rate of reaction Metal 10h Redox cofactors pKa shifters Binding substrates in proper orientation e g cytochromes Mediating oxidation reduction reactions Electrostatically stabilizing Shielding negative charges that would otherwise repel the attack from an electrophile lelectrostatic Metalloenzymes contain tightly bound metal 10ns fet2 e 3 cat zn 2 Metal activated enzymes contain loosely bound metal 10ns Nat KIMg orca 12 Chymotrypsin Mechanism step substrate binding side chain of substrate binds residue adjacent 10 the peptide bond 10 be cleared nestles na chymotrypsin lfreeenzymelhydropnobicpockec ion neenzy me gg ASPposH10nthg hepep 1debondfora tacKAc ivesi e JYH cid 15483 hole Hydrophobic pocket substrate a polypeptide AA E IE Y y E g aa Enzyme substrate complex 1 4 H Hiv Amino acid residues General acid form proton donor Genera base form proton acceptor R COOH R COO Glu Asp Lys Arg Cys His Ser Tyr BINH H H R SH R C CH It HN yNH H R OH R Nitz R S R C CH HN N R O R OH R 0 covalent Change reaction paths Transient bond between the enzyme substrate serine proteases acyl serine intermediate cysteine proteases acyl eystein intermediate protein kinases phosphates phospho amino acid intermediate pyridoxal cont g enzymes lysine 1111 12 Schiff bases changes direct pathway uncatlyzed A B AB catalyzed catalyst A B X A B A 1 0 Requires a nucleophile on the enzyme Donation of electrons from enzyme nucleophile 10 substrate Cys Asp Lys and Ser can participate in side chains of His covalent catalysis by acting as nucleophiles Chymotrypsin Digestion Dietary proteins broken down into small peptides by proteases One of several proteases that cuts peptides at specific locations on peptide backbone Able 10 Cleave the peptide bond adjacent 10 aromatic amino acids Chymotrypsin cuts bond CY fHs 1 Too CHZ H2 CH H cid 15483 cid 15483 cid 15483 cid 15483 YM I step 2 Nucleophilic attack Interaction of Ser nd Hiss a strong nucleophilic alkoxide Lonon Seri group forming a tetrahedral acyl Substrate enzyme accompanied by formation of complex a short lived C charge on the carbonyl generates Aa did TF VHis N 1 i ser d E AA oxygen otthe substrate s iabinzedbsmdr fa II i i FI bonding in the oxyanionhole THIS 1 E a 2 H ser Gly short lived intermediate C acylation H CHZOH 1H H g Ny g 11 I N Amino terminal end Electrostatic Charge step 3 substrate cleavage short lived intermediate tetrahedral acylation Instability of negative charge on the substrate carbonyl oxygen leads 10 Collapse of the intermediates reformation of a double bond with carbon displaces the bond between carbon and the amino group of the peptide linkage breaking the peptide bond Amino leaving group s protonated by Hiss facilitating its displacement a I step water comes In Acy enzyme intermediate Ei cid 15483 Ifi fi s cid 15483 q Acyl enzyme intermediate 4 JVHIS H N AA An incoming water molecule Is deprotonated by general base catalysis generating a strongly nucleophilic hydroxide 10h Attack of hydroxide on the ester linkage of the acyl enzyme generates a second tetrahedral intermediate with oxygen in the oxyamon naeagam iaangor ae charge steps Water Attacks short lived intermediate ldeacylatlon y E H AA CH C 19 JYH s Fyi se H Gly g H o F THIS r I 11 N AA CH CH t 11 19 l se H Gly Collapse of tetrahedral intermediate forms second product carboxylate Anton displaces Ser ag Product H c AA Acy enzyme intermediate Frith aa 1 1 u H 5 Step 6 Break off from the Enzyme Enzyme product 2 Complex His N 1 HH H 0 AA 1 49 ser any short lived intermediate cid 15483 cid 15483 cid 15483 9 9 I 71s AA at j ser H Gly Collapse of tetrahedral intermediate forms second product carboxylate anion displaces seria step Product Dissociates chymotrypsin Cfree enzymes at THIS Active site in oxyanion hole M i Yser Hydrophobic pocket o ly Product 2 Enzyme product 2 complex a a EH HN H H s Hyser G I DISSOCIATION of second product from the active site regenerates free enzyme CLEFT oxy anion 7 FG sauna a g N Activated Ser OH attacks C 0 of 2 0 0 o H HH oft g H 0 Tri H hole Tetrahedral intermediate Intermediate Rearranges Chymotrypsin catalysts in on air 0102 0 HO 1 Catyl 11C Triad Uncompleted enzyme 0M n ago 7 H Ho E tbh Record Product Departs o o H H A 1 N N HH wit O of 6 0 hi Oxy anton Tetrahedral Intermediate Intermediate Rearranges 0 HE 0 H 7 1 H By OF IF Water Binds and Attacks 0 0 CU of 1 1 o HN 4 c Acyl First Product Departs

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