BIOL 302 1st Edition Lecture 13Outline of Last Lecture I. Two Substrate ReactionsII. Proximity and orientation effectsIII. Preferential binding of the transition state complexIV. Catalysis by destabilizing the substrateV. Acid-base catalysisVI. Covalent catalysis (electrophilic and nucleophilic catalysis)VII. Metal ion catalysisOutline of Current Lecture I. Chymotrypsin reaction mechanismII. ProteasesCurrent LectureI. ChymotrypsinA. Substrate Specificity: Cleavage occurs at the C-terminal side of aromatic and largehydrophobic residues1. Ping-Pong Kinetics2. Nucleophilic Catalysis3. Stabilization of the transition state4. General Acid/Base CatalysisII. Chymotrypsin, Trypsin, Elastase,SubtilisinA. catalytic triad of serin proteasesThese notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.III. Specificity pockets in serine protease define specific cleavage sitesIV. ChymotrypsinA. Reaction Catalyzed1. Favorable ∆G2. But very high ∆G‡V. Chymotrypsin AssayA. product formationVI. Inhibition by DIPFA. evidence for nucleophilic catalysisVII. Burst kineticsA. evidence for nucleophilic catalysisVIII. ChymotrypsinA. Reaction mechanismIX. Reaction mechanismA. First, substrate binds to enzyme with peptide bond to be hydrolyzed close to the –O- of SerB. Substrate in redX. Reaction mechanismA. Nucleophilic attack by serine at peptide backbone. Negative charge stabilized by two H-bonds to backbone –NH on Gly193 and Ser195.B. Which group is functioning as the general base catalyst? XI. Reaction mechanismA. Collapse of tetrahedral intermediate, releasing C-terminal portion of substrate. Assisted by protonation of the amino group.B. Which group is functioning as the general acid catalyst? XII. Reaction mechanismXXIII. Reaction mechanismA. Second reaction, now with water as nucleophileB. Asp, working through His, activates water via general base catalysis.C. Oxygen atom of water attacks carbonyl carbon of acyl-enzymeXIV. Reaction mechanismA. Tetrahedral intermediate stabilized by the same residues in theoxyanion hole.XV. Reaction mechanismA. Tetrahedral intermediate collapses, liberating the O atom of Ser195.B. -O by His makes for good leaving group so again illustrates general acid catalysisXVI. Reaction mechanismA. Enzyme returns to initial stateB. Second product is releasedXVII. Reaction mechanismXVIII. Clicker question: Which role does Ser195 of the catalytic triad of Chymotrypsin play during catalysis?A. General acidB. General base C. NucleophilD. ElectrophilE. Stabilization of transition stateXIX. Clicker question: Which role does Histidine 57 of the catalytic triad play during catalysis in Chymotrypsin?A. General acid catalysisB. General base catalysisC. NucleophilD. ElectrophilE. Stabilization of transition stateXX. Clicker question: Which functional group facilitates a nucleophilic attack on the acyl-enzyme state (where the peptide is covalently bound to the enzyme) and facilitates thereby the conversion to the tetrahedral intermediate?A. OH group of serine 195B. NH group of histidine 57C. COO- group of aspartate 102D. OH group of H2O E. CO-OR group of covalently bound peptideXXI. Site-directed mutagenesis to unravel the function of catalytic residuesXXII. Clicker question: Which amino acid residue is the nucleophile?A. HisB. CysC. SubstrateD. waterE. neither XXIII. Clicker question: What is the role of the Histidine?A. nucleophilic catalystB. electrophilic catalystC. general acid catalystD. general base catalystE. Stabilization of transition state XXIV. Mechanisms of other proteasesA. Nature has evolved multiple independent solutions to the problem of peptide bond cleavageXXV. Regulatory Strategies for ProteasesXXVI. ZymogensA. additional means of regulating
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