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U-M CHEM 451 - Enzyme Catalysis

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Enzyme Catalysis: General OverviewAcid-base catalysisFirst example: keto-enol tautomerizationExample: RNAse ACatalysis by preferential binding of the transition state (shape complementarity)Example: LysozymeCatalytic Antibodies (Abzymes)Related: Electrostatic CatalysisBut these questions are still controversial!Metal ion catalysis: Substitute for ProtonsCovalent CatalysisExample: Formation of Schiff base enhances electrophilicityCoupled reactions can overcome an unfavorable ΔGChapter 3 SummaryCHEM 451 1st Edition Lecture 10-11Outline of Last Lecture I. Forms of Inhibitiona. Lineweaver-Burk PlotsII. Cleland NomenclatureIII. Sequential MechanismsIV. Ping Pong MechanismsOutline of Current Lecture V. Enzyme catalyst overviewVI. Acid-base catalysisa. General: keto-enol tautomerizationb. Example: RNAse AVII. Preferential binding of the transition statea. Example: lysozymeVIII. Catalytic antibodies (abzymes)IX. Electrostatic Catalysisa. Discussion of controversyX. Metal ion catalysisXI. Covalent CatalysisXII. Chapter 3 SummaryCurrent LectureEnzyme Catalysis: General OverviewEnzymes use a limited number of specific catalytic strategies:- Acid-base catalysis- Preferential binding of the transition state: enzymes lower the energy barrier by stabilizing the transition state- Electrostatic catalysis: changes in charge on the substrate while it undergoes catalysis; enzymes can place specific charges for favorable reactions- Metal ion catalysts: (specialized form of electrostatic catalysis); holds a large density of positive charge; proximity effect/orientationThese 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.- Covalent catalysis: make a bond between a substrate and enzyme directly; enzyme is transiently chemically modifiedAcid-base catalysisFirst example: keto-enol tautomerization- Ketones have two stable forms: C=O as well as C=C. C=O (ketone) is electrostatically and thermodynamically favored, but are in equilibrium with the enol form- Enzymes oftentimes invoke enols as intermediates in order to activate carbons in subsequent reactions- How can acids catalyze this reaction?o A nearby acid can donate a proton to the oxygen of the carbonyl, and the methyl group may simultaneously lose a protono Transition state: protonated oxygeno Acid is reformed by water protonation – so water is directly involved here- How do bases catalyze this reaction? o Deprotonates the methyl groupo Water protonates the negative oxygen of the carbonyl- IN THE LAB: Add acid or base to the solution to accelerate the reaction.o But you can only increase the acid or base concentration in solution by so much.o We are limited to how much we can accelerate the reaction – we cannot add both the acid and the base together because they would neutralize.- ENZYMES can carry acids and bases simultaneously!o Form a complex 3D structure, positioning specific catalysts close to the substrate in places where they can act separately.o Acids and bases will not neutralize each other, as they are held in place.Example: RNAse A- Cleavage reaction in a long chain of RNA molecules- Place a base Histidine 12 near a 2’OH to deprotonate, activating it as a nucleophile- O- attacks phosphorus, placing a negative charge on phosphate.- Phosphate is reprotonated by acid Histidine 119 placed nearby- Recycling of proton/acid and base histidines so that enzyme can turn over next substrateCatalysis by preferential binding of the transition state (shape complementarity)- Mechanism: binding the transition state to an enzyme with greater affinity than the corresponding substrates or productso NOTE: binding itself is not catalysis.- Rack mechanism: enzymes mechanically strain their substrates toward the transition state geometry through binding sites into which undistorted substrates did not properly fit. o The strained reactant more closely resembles the transition state of the reaction than does the corresponding unstrained reaction. - Interactions that preferentially bind the transition state increase its concentration, proportionally increasing the reaction rate. Example: Lysozyme- Lysozyme degrades cell wall of bacteria by hydrolyzing the beta-1,4 glycosidic linkages from N-acetylmuramic acid (NAM) to N-acetylglucosamine (NAG) – first defense response in eyes- Enzyme is covalently linked to half of the product- Reaction is strongly inhibited by N-lactam cyclic ester (competitive inhibition)o Substrate is sp3 hybridized; inhibitor is sp2o This is a transition state analog – n-lactam more closely resembles the transition state intermediate than the substrate.- ES transition state complex is lower in energy than ES complex, lowering the reaction barrier because it binds the transition state more thermodynamically tightly than the substrate.Catalytic Antibodies (Abzymes)- When bacteria enters body, B-cell encounters antigen. Exposes surface on outside and is activated. B-cell is activated and begins cell division, protecting you from further attack.- B-cells proliferate, precipitating antigen out of body, macrophages come clean it out- To immunize, inject a chemically stable molecule that resembles transition state analog in order to create catalytic antibody, which is potent in catalyzing the immune responseo Claisen rearrangement from chorismate to prephenateo Transition state undergoes cyclo-addition reactiono Transition state has an analog that is chemically similar: bi-cyclico Make antibody the same way as lysozyme above.- Compare to E. coli: naturally creates antibodies with even better hydrogen-bonding stabilization.o Binding affinity of abzymes much lower than true enzymeso Transition state binding has only a limited catalytic effect.Related: Electrostatic Catalysis- Example: lysozyme transition state; negative charge is moving toward oxygen because C=O bond is brokeno Carboxylate group of aspartate attacks carbon, pushing out electrons toward oxygen – ELECTROSTATIC REPULSIONo Proton: where electrons of leaving group want to be – ELECTROSTATIC ATTRACTION- Ketosteroid isomerase (KSI)o Negative charge must be concentrated on oxygeno Place positive charge (protonated carboxyl group), form hydrogen bond around oxygen, stabilizing the O-. o Phenolate: easier to analyze this reaction.o Makes it either easier or harder for oxygen to accumulate negative charge- How important is it for the oxygen to accumulate negative charge?o


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