Berg Tymoczko Stryer Biochemistry Seventh Edition Chapter 9 Catalytic Strategies Copyright 2012 by W H Freeman and Company Concepts Mechanisms used by enzymes to increase catalytic rates include Binding mechanisms Reactive cleft Induce fit Strong binding to transition states Chemical mechanisms Covalent catalysis Acid base catalysis Metal ion catalysis Examples of catalytic strategies Chymotrypsin protease catalytic strategies Catalytic triad Acyl enzyme intermediate Carbonic anhydrases catalytic strategies Zinc assisted activation of H2O Proton shuttle and effect of buffer Binding mechanisms Binding of the substrate to enzyme by multiple weak interactions provides free energy for high interaction specificity and increased catalytic efficiency Reactive cleft provides an environment that favor interaction between S and E close proximity of substrate to active reactive groups optimized orientation of substrate for reaction reaction is protected from H20 so no risk of hydrolysis of intermediates Induced fit stabilizes various conformations for both substrate and enzyme optimized orientation of catalytic groups in enzyme allows very tight binding to transition state Tight binding to transition state provides free energy to accelerate catalysis General mechanisms seen for most enzymes Chemical mechanisms Different enzymes use different chemical mechanisms to catalyze reactions 1 Covalent catalysis Some reactive groups of the enzyme become covalently attached transiently to the substrate Covalent enzyme substrate bond highly reactive for the next reaction step Generally a strong nucleophile group is involved electron rich group attracting nuclei unprotonated imidazole in Histidine unprotonated amine of Lysine Hydroxyl group of Serine Thiolate anion S of Cysteine unprotonated carboxylate of Glutamic acid or Aspartic acid 2 Acid base catalysis Reactive groups of the enzyme capable of donating a proton general acid accepting a proton general base Histidine amino groups Cysteine Lysine Arginine Glutamic acid Aspartic acid Chemical mechanisms continued 3 Metal ion catalysis Either loosely bound to enzyme associate and dissociate e g Mg2 Ca2 Tightly bound prosthetic groups e g Fe2 Zn2 Ionic interactions with substrate or enzyme groups Can shield negative charges or stabilize transient charges on substrate or on the transition state Protease Chymotrypsin as an example of chemical enzymatic mechanisms Protease from the pancreas involved in protein breakdown Catalyzes the hydrolysis of peptide bonds in proteins Cuts after large aa Tryptophan Phenylalanine Methionine Chemical mechanisms of chymotrypsin catalysis Chymotrypsin is a serine protease It uses Ser 195 residue as a highly reactive group for catalysis through a transient covalent interaction with the backbone of the cleaved protein Catalysis in 2 phases 1 acylation phase 1 and 2 deacylation phase 2 An acyl enzyme intermediate is formed between the two phases Acyl group Acyl enzyme intermediate O II CII R O II SerI CII R O Phase 1 Acylation Phase 2 Deacylation Chemical mechanisms of chymotrypsin catalysis continued Phase 1 Acylation Ser 195 is made highly nucleophile Ser OH group attacks the carbonyl C on substrate covalent bond formed Amine part of the cleaved protein blue X is released Phase 2 Deacylation A H2O molecule 2nd substrate carries nucleophilic attack on the carboxylate ester of acyl enzyme regenerate hydroxyl group Ser OH group and carboxyl terminus on cleaved protein Catalytic triad in chymotrypsin catalysis An hydrogen bounded network is formed between Asp 102 His 57 Ser 195 in the catalytic site to orient Ser 195 and make it highly nucleophilic In the presence of substrate conformational changes in the catalytic pocket that lead to 1 His 57 accept a proton from OH Ser 195 general base mechanism 2 The NH group of His 57 is hydrogen bonded to Asp 102 Help orientation of His and Ser and stabilizes HisH 3 Oxygen atom OH Ser activated into alkoxide ion now poised for nucleophilic attack of the protein substrate Catalytic triad is found in many other peptide cleaving enzymes from different species highly evolved catalytic mechanism Peptide hydrolysis by chymotrypsin Orientation and Nucleophilic attack Stabilization of transition state and formation of intermediate 1 Substrate binding 1 2 Stabilization by oxyanion hole Oxyanion hole area of the active catalytic site that tightly bind tetrahedral transition state intermediates Hydrogen bonds formed between peptide backbone of the enzyme and oxygen atom derived from the carbonyl group of activated substrate Stabilizes the charge of negative oxygen atom oxyanion Interaction could not have happen with carbonyl oxygen C O but possible with longer C 0 bond in tetrahedral intermediate Peptide hydrolysis by chymotrypsin Orientation and Nucleophilic attack Stabilization of transition state and formation of intermediate 1 Break of peptide bond and covalent E S bond 1 Release of amine product End step 1 Substrate binding Enzyme reset ready for another round Release of carboxyl product End step 2 Break of covalent E S bond transition state and Stabilization of formation of intermediate 2 2 Nucleophilic attack of H2O on carboxylate ester of acyl E Substrate specificity of chymotrypsin Chymotrypsin cut after bulky and hydrophobic aa residues Other proteases with a similar structure and the same catalytic mechanisms cut after different set of aa e g Trypsin cuts after long positively charge aa Lys Arg Elastase cuts after aa with small side chains Ala Ser Where does this difference in specificity comes from Chymotrypsin binding pocket is deep and hydrodrophobic Trypsin pocket has Asp at the bottom Elastase pocket is narrow Site directed mutagenesis reveal catalytic triad efficiency Mutating key amino acids in the catalytic triad allows for an effective dissection of its importance for protease catalytic activity e g Subtilisin a bacterial protease uses Asp 32 His 64 Ser 221 to cut peptide bonds Mutations of the three amino acids in catalytic triad to alanine lead to dramatic decrease in catalytic efficiency Reduction by more than a million fold 106 Alternatives to Serine for proteolytic activity Some proteases do not use Ser to cut peptide bonds Other major classes include Cys proteases e g caspases also have a catalytic mechanism that resemble the triad of Ser proteases Asp proteases e g pepsin renin use Asp carboxylate group to activate H2O and attack peptide bonds Metalloproteases e g