lehninger ppt ch06 - class...
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6| Enzymes
© 2013 W. H. Freeman and Company
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CHAPTER 6 Enzymes
– Physiological significance of enzymes– Origin of catalytic power of enzymes– Chemical mechanisms of catalysis– Mechanisms of chymotrypsin and lysozyme– Description of enzyme kinetics and inhibition
Key topics about enzyme function:
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What are enzymes?
•Enzymes are catalysts• Increase reaction rates without being used up
•Most enzymes are globular proteins • However, some RNA (ribozymes and ribosomal RNA) also
catalyze reactions
• Study of enzymatic processes is the oldest field of biochemistry, dating back to late 1700s
• Study of enzymes has dominated biochemistry in the past and continues to do so
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Why biocatalysis over inorganic catalysts?• Greater reaction specificity: avoids side products• Milder reaction conditions: conducive to conditions in cells• Higher reaction rates: in a biologically useful timeframe
• Capacity for regulation: control of biological pathways
COO
OHO COO
COO
O COO
NH2
OOCCOO
O
OH
OH
COO
NH2
COO
-
-
-
-
-
-
--
Chorismate mutase
• Metabolites have many potential pathways of decomposition
• Enzymes make the desired one most favorable
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Enzymatic Substrate Selectivity
No binding
OOC NH3
H
OOC NH3
H
H NH
HOH
OH
H
OH
CH3
OOC NH3
HOH
--
-
+
+
+
Binding but no reaction
Example: Phenylalanine hydroxylase
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Reaction Conditions Compatible with Life
37˚C
pH ≈7
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Six Classes of Enzymes:Defined by the Reactions Catalyzed
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Enzyme‐Substrate Complex
• Enzymes act by binding substrates– The noncovalent enzyme substrate complex is known as the Michaelis complex
– Description of chemical interactions– Development of kinetic equations
][]][[
SKSEkv
m
cat
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Enzyme‐Substrate Complex
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Enzymatic Catalysis
• Enzymes do not affect equilibrium (ΔG)• Slow reactions face significant activation barriers (ΔG‡) that must be surmounted during the reaction
• Enzymes increase reaction rates (k) by decreasing ΔG‡
k kBTh
exp
G
RT
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Reaction Coordinate Diagram
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Enzymes Decrease ΔG‡
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Rate Enhancement by Enzymes
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How to Lower G
Enzymes organize reactive groups into close proximity and proper orientation
• Uncatalyzed bimolecular reactions two free reactants single restricted transition state conversion is entropically unfavorable
• Uncatalyzed unimolecular reactions flexible reactant rigid transition state conversion is entropically unfavorable for flexible reactants
• Catalyzed reactionsEnzyme uses the binding energy of substrates to organize the reactants to a fairly rigid ES complexEntropy cost is paid during bindingRigid reactant complex transition state conversion is entropically OK
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Support for the Proximity ModelThe rate of anhydride formation from esters and carboxylates shows a strong dependence on proximity of two reactive groups (work by Thomas C. Bruice’s group).
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How to Lower G
Enzymes bind transition states best
• The idea was proposed by Linus Pauling in 1946– Enzyme active sites are complimentary to the transition state of the reaction
– Enzymes bind transition states better than substrates– Stronger/additional interactions with the transition state as compared to the ground state lower the activation barrier
Largely ΔH‡ effect
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Illustration of TS Stabilization Idea:Imaginary Stickase
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Catalytic Mechanisms
– acid‐base catalysis: give and take protons– covalent catalysis: change reaction paths– metal ion catalysis: use redox cofactors, pKa shifters– electrostatic catalysis: preferential interactions with TS
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General Acid‐Base Catalysis
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Amino Acids in General Acid‐Base Catalysis
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Covalent Catalysis
• A transient covalent bond between the enzyme and the substrate
• Changes the reaction Pathway
– Uncatalyzed:
– Catalyzed:
• Requires a nucleophile on the enzyme– Can be a reactive serine, thiolate, amine, orcarboxylate
B A B—AOH2
B :X A B X—A :X B—AOH2
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Metal Ion Catalysis
• Involves a metal ion bound to the enzyme
• Interacts with substrate to facilitate binding– Stabilizes negative charges
• Participates in oxidation reactions
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Chymotrypsin uses most of the enzymatic mechanisms
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Active Site of Chymotrypsin with Substrate
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Chymotrypsin Mechanism Step 1: Substrate Binding
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Chymotrypsin MechanismStep 2: Nucleophilic Attack
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Chymotrypsin MechanismStep 3: Substrate Cleavage
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Chymotrypsin MechanismStep 4: Water Comes In
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Chymotrypsin MechanismStep 5: Water Attacks
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Chymotrypsin MechanismStep 6: Break‐off from the Enzyme
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Chymotrypsin MechanismStep 7: Product Dissociates
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Peptidoglycan and Lysozyme
• Peptidoglycan is a polysaccharide found in many bacterial cell walls
• Cleavage of the cell wall leads to the lysis of bacteria
• Lysozyme is an antibacterial enzyme
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Peptidoglycan and Lysozyme
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X‐ray structures of lysozyme with bound substrate analogs show that the C‐1 carbon is located between Glu 35 and Asp 52 residues.
General Acid‐Base + Covalent Catalysis: Cleavage of Peptidoglycan by Lysozyme
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Cleavage of Peptidoglycan by Lysozyme: Two Successive SN2 Steps Model
• Asp 52 acts as a nucleophile to attack the anomeric carbon in the first SN2 step
• Glu 35 acts as a general acid and protonates the leaving group in the transition state
• Water hydrolyzes the covalent glycosyl‐enzyme intermediate
• Glu 35 acts as a general base to deprotonate water in the second SN2 step
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What is enzyme kinetics?
• Kinetics is the study of the rate at which compounds react
• Rate of enzymatic reaction is affected by:– enzyme– substrate– effectors– temperature
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Why study enzyme kinetics?
• Quantitative description of biocatalysis• Determine the order of binding of substrates• Elucidate acid‐base catalysis• Understand catalytic mechanism• Find effective inhibitors• Understand regulation of activity
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How to Do Kinetic MeasurementsExperiment:1) Mix enzyme + substrate2) Record rate of substrate disappearance/product formation as a function of time
(the velocity of reaction)3) Plot initial velocity versus substrate concentration.4) Change substrate concentration and repeat
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Effect of Substrate Concentration
• Ideal rate:
• Deviations due to:– limitation of measurements– substrate inhibition– substrate prep contains inhibitors– enzyme prep contains inhibitors
SKSVv
m
][max
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Effect of Substrate Concentration
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Saturation Kinetics: At high [S] velocity does not depend on [S]
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Determination of Kinetic Parameters
Nonlinear Michaelis‐Menten plot should be used to calculate parameters Km and Vmax.
Linearized double‐reciprocal plot is good for analysis of two‐substrate data or inhibition.
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Lineweaver‐Burk Plot:Linearized, Double‐Reciprocal
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Derivation of Enzyme Kinetics Equations
• Start with a model mechanism• Identify constraints and assumptions• Carry out algebra ...
– ... or graph theory for complex reactions
• Simplest Model Mechanism: E + S ES E + P– One reactant, one product, no inhibitors
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Identify Constraints and Assumptions
• Total enzyme concentration is constant– Mass balance equation for enzyme: ETot = [E] + [ES]– It is also implicitly assumed that: STot = [S] + [ES] ≈ [S]
• Steady state assumption
• What is the observed rate?– Rate of product formation
vnet dPdt
k[ES]
0ESofbreakdownofrateESofformationofrate][
dtESd
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Carry out the algebra• The final form in case of a single substrate is
• kcat (turnover number): how many substrate molecules can one enzyme molecule convert per second
• Km (Michaelis constant): an approximate measure of substrate’s affinity for enzyme
• Microscopic meaning of Km and kcat depends on the details of the mechanism
][]][[
SKSEkv
m
totcat
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Enzyme efficiency is limited by diffusion:kcat/KM
• Can gain efficiency by having high velocity or affinity for substrate– Catalase vs. acetylcholinesterase
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Two‐Substrate Reactions
•Kinetic mechanism: the order of binding of substrates and release of products
•When two or more reactants are involved, enzyme kinetics allows to distinguish between different kinetic mechanisms– Sequential mechanism– Ping‐Pong mechanism
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Sequential Kinetic Mechanism• We cannot easily distinguish random from ordered• Random mechanisms in equilibrium will give intersection point at y‐axis
• Lineweaver‐Burk: lines intersect
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Ping‐Pong Kinetic MechanismLineweaver‐Burk: lines are parallel
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Enzyme InhibitionInhibitors are compounds that decrease enzyme’s activity
• Irreversible inhibitors (inactivators) react with the enzyme• One inhibitor molecule can permanently shut off one enzyme molecule• They are often powerful toxins but also may be used as drugs
• Reversible inhibitors bind to and can dissociate from the enzyme• They are often structural analogs of substrates or products• They are often used as drugs to slow down a specific enzyme
• Reversible inhibitor can bind: • to the free enzyme and prevent the binding of the substrate• to the enzyme‐substrate complex and prevent the reaction
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• Competes with substrate for binding – Binds active site– Does not affect catalysis
• No change in Vmax; apparent increase in KM• Lineweaver‐Burk: lines intersect at the y‐axis
Competitive Inhibition
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Competitive Inhibition
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Competitive Inhibition
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Uncompetitive Inhibition
• Only binds to ES complex • Does not affect substrate binding• Inhibits catalytic function
• Decrease in Vmax; apparent decrease in KM• No change in KM/Vmax• Lineweaver‐Burk: lines are parallel
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Uncompetitive Inhibition
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Uncompetitive Inhibition
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Mixed Inhibition
• Binds enzyme with or without substrate― Binds to regulatory site― Inhibits both substrate binding and catalysis
• Decrease in Vmax; apparent change in KM• Lineweaver‐Burk: lines intersect left from the y‐axis• Noncompetitive inhibitors are mixed inhibitors such that there is no change in KM
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Mixed Inhibition
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Mixed Inhibition
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Enzyme activity can be regulated
• Regulation can be:– noncovalent modification– covalent modification– irreversible– reversible
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Noncovalent Modification: Allosteric Regulators
The kinetics of allosteric regulators differ from Michaelis‐Menten kinetics.
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Some Reversible Covalent Modifications
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Zymogens are activated by irreversible covalent modification
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The blood coagulation cascade uses irreversible covalent modification
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Some enzymes use multiple types of regulation
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Chapter 6: Summary
• why nature needs enzyme catalysis• how enzymes can accelerate chemical reactions• how chymotrypsin breaks down peptide bonds• how to perform and analyze kinetic studies• how to characterize enzyme inhibitors • how enzyme activity can be regulated
In this chapter, we learned: