Download Chapter 6 Mechanisms of Enzymes

Horton • Moran • Scrimgeour • Perry • Rawn
Principles of Biochemistry
Fourth Edition
Chapter 6
Mechanisms of Enzymes
Prentice Hall c2002
Chapter 6 Copyright © 2006 Pearson Prentice
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Hall, Inc.
Chapter 6 Mechanisms of Enzymes
• Mechanisms - the molecular details of
catalyzed reactions
• Enzyme mechanisms deduced from:
Kinetic experiments
Protein structural studies
Studies of nonenzymatic model systems
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6.1 The Terminology of Mechanistic Chemistry
• A reaction mechanism describes the details of a
reaction
• Reactants, products and intermediates are
identified
• Enzymatic mechanisms use the same
symbolism used in organic chemistry
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Nucleophilic substitution reactions
• Nucleophilic species are electron rich, and
electrophilic species are electron poor
• Types of nucleophilic substitution reactions
include:
(1) Formation of a tetrahedral intermediate by
nucleophilic substitution
(2) Direct displacement via a transition state
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Two types of nucleophilic
substitution reactions
• Formation of a tetrahedral intermediate
• Direct displacement
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Cleavage reactions
• Carbanion formation
• Carbocation formation
• Free radical formation
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Oxidation-reduction reactions
• Electrons are transferred between two species
• Oxidizing agent gains electrons (is reduced)
• Reducing agent donates electrons (is oxidized)
• Formaldehyde is oxidized by oxygen (below)
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6.2 Catalysts Stabilize Transition States
Fig 6.1 Energy
diagram for a singlestep reaction
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Fig 6.2 Energy diagram for
reaction with intermediate
• Intermediate occurs in
the trough between the
two transition states
• Rate determining step
in the forward direction
is formation of the first
transition state
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Enzymes lower the activation
energy of a reaction
(1) Substrate binding
• Enzymes properly position substrates for reaction
(makes the formation of the transition state more
frequent and lowers the energy of activation)
(2) Transition state binding
• Transition states are bound more tightly than
substrates (this also lowers the activation energy)
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Fig 6.3 Enzymatic catalysis of the reaction
A+B
A-B
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6.3 Chemical Modes of Enzymatic Catalysis
A. Polar Amino Acid Residues in Active Sites
• Active-site cavity of an enzyme is lined with
hydrophobic amino acids
• Polar, ionizable residues at the active site
participate in the mechanism
• Anions and cations of certain amino acids are
commonly involved in catalysis
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Table 6.1
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Table 6.2 pKa Values of amino acid
ionizable groups in proteins
Group
Terminal a-carboxyl
Side-chain carboxyl
Imidazole
Terminal a-amino
Thiol
Phenol
pKa
3-4
4-5
6-7
7.5-9
8-9.5
9.5-10
e-Amino
~10
Guanidine
~12
Hydroxymethyl
~16
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B. Acid-Base Catalysis
• Reaction acceleration is achieved by catalytic
transfer of a proton
• A general base (B:) can act as a proton acceptor
to remove protons from OH, NH, CH or other XH
• This produces a stronger nucleophilic reactant (X:-)
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General base catalysis reactions (continued)
• A general base (B:) can remove a proton from
water and thereby generate the equivalent of
OH- in neutral solution
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Proton donors can also catalyze reactions
• A general acid (BH+) can donate protons
• A covalent bond may break more easily if one
of its atoms is protonated (below)
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C. Covalent Catalysis
• All or part of a substrate is bound covalently to
the enzyme to form a reactive intermediate
• Group X can be transferred from A-X to B in two
steps via the covalent ES complex X-E
A-X + E
X-E + A
X-E + B
B-X + E
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Sucrose phosphorylase exhibits
covalent catalysis
Step one: a glucosyl residue is transferred to enzyme
*Sucrose + Enz
Glucosyl-Enz + Fructose
Step two: Glucose is donated to phosphate
Glucosyl-Enz + Pi
Glucose 1-phosphate + Enz
*(Sucrose is composed of a glucose and a fructose)
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D. pH Affects Enzymatic Rates
• Catalytic mechanisms are dependent upon the
state of ionization of catalytic groups
• Plots of reaction velocity versus pH can
indicate important ionizable catalytic groups
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Fig 6.4 pH-rate profile for papain
• The two
inflection points
approximate the
pKa values of
the two ionizable
residues
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Fig 6.5
• Papain’s activity depends
upon ionizable residues:
His-159 and Cys-25
(a) Ribbon model
(b) Active site residues
(N blue, S yellow)
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Fig. 6.6
Three ionic forms
of papain. Only the
upper tautomer of
the middle pair is
active
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6.4 Diffusion-Controlled Reactions
• Enzyme rates can approach the physical limit of
the rate of diffusion of two molecules in solution
• Under physiological conditions the encounter
frequency is about 108 to 109 M-1s-1
• A few enzymes have rate-determining steps that
are roughly as fast as the binding of substrates to
the enzymes
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Table 6.4
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A. Triose Phosphate Isomerase (TPI)
• TPI catalyzes a rapid aldehyde-ketone
interconversion
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Role of active site residues in TPI catalysis
• TPI has two ionizable active-site residues
• Glu acts as a general acid-base catalyst
• His shuttles protons between oxygens of an
enzyme-bound intermediate
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Fig 6.7 Proposed mechanism for TPI
• General acid-base catalysis mechanism (4 slides)
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Fig 6.7 TPI mechanism (continued)
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Fig 6.7 TPI mechanism (continued)
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Fig 6.7 TPI mechanism (continued)
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Fig 6.9 Energy diagram for the TPI reaction
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B. Superoxide Dismutase
• Superoxide dismutase is a very fast catalyst
because:
(1) The negatively charged substrate is
attracted by a positively charged electric
field near the active site
(2) The reaction itself is very fast
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Two step reaction of superoxide dismutase
• An atom of copper bound to the
enzyme is reduced and then oxidized
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6.5 Binding Modes of Enzymatic Catalysis
• Proper binding of reactants in enzyme active sites
provides substrate specificity and catalytic power
• Two catalytic modes based on binding properties
can each increase reaction rates over 10,000-fold :
(1) Proximity effect - collecting and positioning
substrate molecules in the active site
(2) Transition-state (TS) stabilization - transition
states bind more tightly than substrates
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Binding forces utilized for catalysis
1. Charge-charge interactions
2. Hydrogen bonds
3. Hydrophobic interactions
4. Van der Waals forces
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A. The Proximity Effect
• Correct positioning of two reacting groups
(in model reactions or at enzyme active sites):
(1) Reduces their degrees of freedom
(2) Results in a large loss of entropy
(3) The relative enhanced concentration of
substrates (“effective molarity”) predicts the rate
acceleration expected due to this effect
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Effective molarity
k1(s-1)
Effective molarity =
k2(M-1s-1)
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Fig 6.11 Reactions of carboxylates
with phenyl esters
• Increased rates are seen when the reactants are held more
rigidly in proximity (continued next slide)
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Fig. 6.11 (continued)
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B. Weak Binding of Substrates to Enzymes
• Energy is required to reach the transition state
from the ES complex
• Excessive ES stabilization would create a
“thermodynamic pit” and mean little or no catalysis
• Most Km values (substrate dissociation constants)
indicate weak binding to enzymes
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Fig 6.12 Energy of substrate binding
• If an enzyme
binds the
substrate too
tightly (dashed
profile), the
activation barrier
(2) could be
similar to that of
the uncatalyzed
reaction (1)
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C. Induced Fit
• Induced fit activates an enzyme by substrateinitiated conformation effect
• Induced fit is a substrate specificity effect, not a
catalytic mode
• Hexokinase mechanism requires sugar-induced
closure of the active site
Glucose + ATP
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Glucose 6-phosphate + ADP
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Fig 6.13 Stereo views of yeast hexokinase
• Yeast hexokinase
contains 2 domains
connected by a
hinge region.
Domains close on
glucose binding.
(a) Open
conformation
(b) Closed
conformation
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D. Transition-State (TS) Stabilization
• An increased interaction of the enzyme and
substrate occurs in the transition-state (ES‡)
• The enzyme distorts the substrate, forcing it
toward the transition state
• An enzyme must be complementary to the
transition-state in shape and chemical character
• Enzymes may bind their transition states 1010 to
1015 times more tightly than their substrates
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Transition-state (TS) analogs
• Transition-state analogs are stable compounds
whose structures resemble unstable transition states
• Fig. 6.14 2-Phosphoglycolate, a TS analog for the
enzyme triose phosphate isomerase
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Fig 6.15 Inhibition of adenosine
deaminase by a TS analog
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Catalytic antibodies
• Fig 6.16 Antibodycatalyzed amide
hydrolysis
(a) TS analog antigen
(b) Reaction catalyzed
by the catalytic
antibody
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6.6 Lysozyme Binds an Ionic
Intermediate Tightly
• Lysozyme binds polysaccharide substrates (the
sugar in subsite D of lysozyme is distorted into
a half-chair conformation)
• Binding energy from the sugars in the other
subsites provides the energy necessary to
distort sugar D
• Lysozyme binds the distorted transition-state
type structure strongly
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Fig 6.17 Bacterial cell-wall polysaccharide
• Lysozyme cleaves bacterial cell wall polysaccharides
(a four residue portion of a bacterial cell wall with
lysozyme cleavage point is shown below)
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Fig 6.18 Lysozyme from chicken with a
triaccharide molecule (pink)
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Fig 6.19 Conformations of
N-acetylmuramic acid
(a) Chair conformation
(b) D-Site sugar residue
is distorted into a
higher energy halfchair conformation
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Lysozyme reaction mechanisms
1. Proximity effects
2. Acid-base catalysis
3. TS stabilization (or substrate distortion
toward the transition state)
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Fig 6.20 Mechanism of lysozyme (2 slides)
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6.7 Properties of Serine Proteases
A. Zymogens Are Inactive Enzyme Precursors
• Digestive serine proteases including trypsin,
chymotrypsin, and elastase are synthesized
and stored in the pancreas as zymogens
• Storage of hydrolytic enzymes as zymogens
prevents damage to cell proteins
• Zymogens are activated by selective proteolysis
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Fig 6.21 Activation of some
pancreatic zymogens
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Fig 6.22 Stereo view of the backbones of
chymotrypsinogen and a-chymotrypsin
Chymotrypsinogen (blue),
a-chymotrypsin (green)
Catalytic Asp, His, Ser
(red), Ile-16, Asp-194
(yellow)
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B. Substrate Specificity of Serine Proteases
• Many digestive proteases share similarities in
1o,2o and 3o structure
• Chymotrypsin, trypsin and elastase have a
similar backbone structure
• Active site substrate specificities differ due to
relatively small differences in specificity pockets
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Fig 6.23 Comparison of the backbones of (a)
chymotrypsin (b) trypsin and (c) elastase
• Backbone conformations and active-site residues
(red) are similar in these three enzymes
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Fig 6.24 Binding sites of chymotrypsin,
trypsin, and elastase
• Substrate
specificities are
due to relatively
small structural
differences in
active-site
binding cavities
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C. Serine Proteases Use Both the Chemical
and Binding Modes of Catalysis
• a-Chymotrypsin active site groups include:
Ser-195 - identified by covalent DFP labeling
His-57 - identified by affinity labeling with
TosPheCH2Cl
Asp-102 - identified by X-ray crystallography
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Fig 6.25 Stereo view of the
catalytic site of chymotrypsin
• Active-site Asp102, His-57, Ser195 are arrayed in
a hydrogenbonded network
(O red, N blue)
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Fig 6.26 Catalytic triad of chymotrypsin
• Imidazole ring (His-57) removes H from Ser-195
hydroxyl to make it a strong nucleophile (-CH2O-)
• Buried carboxylate (Asp-102) stabilizes the positivelycharged His-57 to facilitate serine ionization
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Fig 6.27 a-Chymotrypsin mechanism (8 slides)
Step (1): E + S
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Fig 6.27 (E-S)
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Fig 6.27 (E-TI1)
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Fig 6.27 (Acyl E + P1)
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Fig 6.27 (Acyl E + H2O)
(4)
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Fig 6.27 (E-TI2)
(5)
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Fig 6.27 (E-P2)
(6)
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Fig 6.27 (E + P2)
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