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Catalytic Mechanisms
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To understand how enzymes work at the molecular level.
Objective To understand how enzymes work at the molecular level. Ultimately requires total structure determination, but can learn much through biochemical analysis.
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To Be Explained Specificity Catalysis For specific substrates
Amino acids residues involved Catalysis Mechanisms Amino acids involved/Specific role(s)
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Enzyme Binding Sites Active Site: Regulatory Site:
Substrate Binding Site + Catalytic Site Regulatory Site: a second binding site, Binding by regulatory molecule affects the active site alter the efficiency of catalysis improve or inhibit
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General Characteristics
Three dimensional space Occupies small part of enzyme volume Clefts or crevices Ligands (substrate or effector) bound by multiple weak interactions Specificity depends on precise arrangement of atoms in active site
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Models Induced Fit Lock and Key
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Identification and Characterization of Active Site
Structure: size, shape, charges, etc. Composition: identify amino acids involved in binding and catalysis.
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Binding or Positioning Site (Trypsin)
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Binding or Positioning Site (Chymotrypsin)
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Catalytic Site (e.g. Chymotrypsin)
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Probing the Structure of the Active Site
Model Substrates
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Model Substrates (Chymotrypsin)
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Peptide Chain? All Good Substrates!
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a-amino group? Good Substrate!
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Side Chain Substitutions
Good Substrates t-butyl- Cyclohexyl
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Conclusion Bulky Hydrophobic Binding Site
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Probing the Structure of the Active Site
Competitive Inhibitors
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Arginase
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Good Competitive Inhibitors
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Poor Competitive Inhibitors
All Three Charged Groups are Important
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Conclusion Active Site Structure of Arginase
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Identifying Active Site Amino Acid Residues
Covalent modification of residues Inactivation of enzyme Site directed mutagenesis
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Mechanisms of Catalysis
Acid-base catalysis Covalent catalysis Metal ion catalysis Proximity and orientation effects Preferential binding (stabilization) of the transition state
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Addition or removal of a proton by side chains
Acid-Base Catalysis Addition or removal of a proton by side chains
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General Acids and Bases
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Keto-Enol Tautomerization
Acid-Base Catalysis Keto-Enol Tautomerization
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Uncatalyzed Reaction
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General Acid Catalysis
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General Base Catalysis
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Ribonuclease A Figure 11-10
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Mechanism of RNase A Get specificity, because DNA doesn’t have an OH group on carbon 2. Figure part 1
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Mechanism of RNase A Figure part 2
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Covalent Catalysis (Nucleophilic catalysis) (Principle)
Involves a transient covalent bond between the enzyme and the substrate Usually by the nucleophilic attack of the substrate by the enzyme
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Covalent Catalysis (Principle)
Slow H2O + A–B ——> AOH + BH A-B + E-H ——> E-A + BH E-A + H2O ——> A-OH + E-H Fast NOTE: New Reaction Pathway
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Covelent Catalysis
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The Schiff Base
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Metal Ion Catalysis Charge stabilization Water ionization
Charge shielding
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Metal Ion Catalysis Metalloenzymes: tightly bound metal ions
Catalytically essential Fe2+, Fe3+, Cu2+, Mn2+, and Co2+ Metal-activated enzymes: loosely bound metal ions (from solution or with substrate) Structural metal ions: Na+, K+, and Ca2+ Both: Mg2+ and Zn2+
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Carbonic Anhydrase
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Carbonic Anhydrase
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Proximity and Orientation Effects Rate of a reaction depends on:
Number of collisions Energy of molecules Orientation of molecules Reaction pathway (transition state)
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Proximity V = k[A][B] [A] and [B] = ~13M on enzyme surface
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Biomolecular Reaction of Imidazole with p-Nitrophenylacetate (Intermolecular)
Page 336
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Intramolecular Rate = 24x Intermolecular Rate
Intramolecular Reaction of Imidazole with p-Nitrophenylacetate (Intramolecular) Within an active site imidazole can be in that proximity to p-NP Intramolecular Rate = 24x Intermolecular Rate Page 336
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Orientation
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Geometry of an SN2 Reaction
Figure 11-14
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Preferrential Binding of Reaction Intermediate
Stabilize Transition State Electrostatic stabilization of developing charge Relief of induced bond angle strain Enhancement of weak interactions between enzyme and intermediate. Draw out diagram.
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Steric Strain in Organic Reactions
Reaction Rate: R=CH3 is 315x vs R=H Page 338
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Effect of Preferential Transition State Binding
Figure 11-15
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Transition State Analogs
Powerful Enzyme Inhibitors
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Proline Racemase (planar transition state)
Page 339
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Transition State Analogs of Proline
Binding = 160x versus Proline Page 339
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Chymotrypsin Trypsin Elastase etc.
Serine Proteases Chymotrypsin Trypsin Elastase etc.
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Convergent Evolution
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Substrate Specificity
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Mechanism of Chymotrypsin
p-nitrophenolate
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X-Ray Structure of Bovine Trypsin (Ribbon Diagram)
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Active Site Residues of Chymotrypsin (Catalytic Triad)
Figure 11-26
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Catalytic Mechanism of the Serine Proteases
Catalytic Triad
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Catalytic Mechanism of the Serine Proteases
Figure part 2
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Catalytic Mechanism of the Serine Proteases
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Catalytic Mechanism of the Serine Proteases
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Catalytic Mechanism of the Serine Proteases
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Catalytic Mechanism of the Serine Proteases
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Catalytic Mechanism of the Serine Proteases
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Transition State Stabilization in the Serine Proteases
Figure 11-30a
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Transition State Stabilization in the Serine Proteases
Figure 11-30b
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Mechanism of Chymotrypsin
p-nitrophenolate New Reaction Pathway (versus uncatalyzed reaction)
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