Transcript Presentation

Mechanism of Enzymes
Mechanisms
the molecular details of catalyzed reactions
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
Two types of nucleophilic
substitution reactions
• Direct displacement
Two types of nucleophilic
substitution reactions
• Formation of a tetrahedral intermediate
Cleavage reactions
• Carbanion formation
• Carbocation formation
Cleavage reactions
• Free radical formation
Catalysts Stabilize Transition States
Energy diagram for a
single-step reaction
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
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)
Enzymatic catalysis of the
reaction A+B
A-B
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
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:-)
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
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)
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)
Triose Phosphate Isomerase
(TPI)
• TPI catalyzes a rapid aldehyde-ketone
interconversion
Proposed mechanism for TPI
• General acid-base catalysis mechanism (4 slides)
Proposed mechanism for TPI
Proposed mechanism for TPI
Proposed mechanism for TPI
Energy diagram for the TPI reaction
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
Binding forces utilized for
catalysis
1. Charge-charge interactions
2. Hydrogen bonds
3. Hydrophobic interactions
4. Van der Waals forces
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
Reactions of carboxylates
with phenyl esters
Reactions of carboxylates
with phenyl esters
Reactions of carboxylates
with phenyl esters
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
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)
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
Transition-state (TS) analogs
• Transition-state analogs are stable compounds
whose structures resemble unstable transition states
• 2-Phosphoglycolate, a TS analog for the enzyme
triose phosphate isomerase
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
Glucose 6-phosphate + ADP
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
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)
Conformations of N-acetylmuramic acid
(a) Chair conformation
(b) D-Site sugar residue
is distorted into a
higher energy halfchair conformation
Lysozyme reaction mechanisms
1. Proximity effects
2. Acid-base catalysis
3. TS stabilization (or substrate distortion
toward the transition state)
Mechanism of lysozyme
Properties of Serine Proteases
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
Activation of some
pancreatic zymogens
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
Binding sites of chymotrypsin,
trypsin, and elastase
• Substrate
specificities are
due to relatively
small structural
differences in
active-site
binding cavities
Identification of His at active site
• The irreversible inhibitor (TosPheCH2Cl) binds to
the active-site His residue in serine proteases
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
a-Chymotrypsin mechanism
(Acyl E + H2O)
(E-TI2)
(E-P2)
(E + P2)