Transcript General acid-base catalysis

Enzymes catalysis
Enzyme catalyzed chemical reactions via both
non-covalent and covalent interactions
• Transient chemical reactions (i.e., covalent
interactions) often occur between substrates
and functional groups in the active sites of
enzymes, thus providing an alternative reaction
path.
• Noncovalent interactions between substrates
and enzymes will generate a binding energy
(ΔGB), which will lower the activation energy of
the reactions.
Weak interactions between enzyme and substrate are
optimized in the transition state
• Enzyme and substrate was proposed to
complement each other like “ a lock and the key”
(Emil Fischer, 1894).
• Years later, it was realized that an enzyme
completely complementary to its substrate
would be a very poor enzyme!
• According to the transition state theory, an
enzyme must be complementary to the reaction
transition state of the reactant (Haldane, 1930;
Pauling, 1946).
An effective enzyme must
have its active site
complementary to the
transition state of the reaction.
Activation energy increases!
ES
E-transition state
E+P
The transition state theory of enzyme catalysis has
strong supporting evidences
• The idea of transition-state analogs was
suggested in accordance with the transition
state theory (Pauling, 1940s) and were later
proved to be correct: such analogs bind to
enzymes 102 to 106 times more tightly than
normal substrates.
• The idea of catalytic antibodies was also
suggested by this theory (Jencks, 1969) and
was proved to be correct later (Lerner and
Schultz, 1980s).
Transition-state
Catalytic antibodies
Transition-state
analog
Catalytic antibodies
Transition-state
analog
Transition-state analogs can be
designed according to the
proposed reaction mechanism
and used as antigens for making
catalytic antibodies.
Transition-state
The binding energy
made available by the
noncovalent enzymesubstrate interactions
often provide a major
driving force for enzyme
catalysis.
Enzyme catalysis
I. Rate Acceleration
Enzyme accelerate the rate of reaction:
•
In most case, initial interaction is
noncovalent (ES) making use of
hydrogen bonding, electrostatic,
hyodrophobic and vander Waals
force to effect binding.
•
ES: Catalytic groups are now an
integral part of the same molecule,
the reaction of enzyme bound
substrates will follow first order
rather than second order kinetics.
E + S --> ES --> [EX*] --> EP --> E + P
Enzyme catalysis
II. Binding Energy in Catalysis:
•
Favorable interaction between
the enzyme and substrate result
in a favorable intrinsic binding
energy.
•
Entropy is lost when substrate
binds to the enzyme.
(a) Two entities become one.
(b) Substrate is less able to rotate.
(c) Substrate become more ordered.
•
Weak interactions between the
enzyme and substrate are
optimize and stabilize the
transition state.
E + S --> ES --> [EX*] --> EP --> E + P
(weak) (stronger)
Factors involved in rate acceleration

Desolvation:
•
When substrate binds to the enzyme surrounding water in solution is replaced
by the enzyme. This makes the substrate more reactive by destablizing the
charge on the substrate.
•
Expose a water charged group on the substrate for interaction with the
enzyme.
•
Also lowers the entropy of the substrate (more ordered).
Factors involved in rate acceleration…….

Strain and Distortion:
 When substrate bind to the enzyme, it may induces a conformational change
in the active site to fit to a transition state.
 Frequently, in the transition state, the substrate and the enzyme have slightly
different structure (strain or distortion) and increase the reactivity of the
substrate.
cyclic phosphate ester
Acylic phospodiester
Binding energy can be used for
selecting specific substrates
and overcome the ΔG ‡
• The reduction in entropy of oriented substrates.
• Desolvation of the substrates.
• Distortion of substrates for converting to the
transition state.
• Proper alignment of catalytic function groups
via induced fit (conformational change) in the
enzyme active site.
• The consumption of binding energy in such
processes will help lower the ΔG ‡ , thus
increasing the reaction rate.
Catalytic Strategies
•
Catalysis by approximation
–
•
Covalent catalysis
–
•
The active site contains a reactive group, usually a powerful nucleophile that become
temporarily covalently modified in the course of catalysis.
General acid-base catalysis
–
•
In reactions that include two substrates, the rate is enhanced by bringing the two
substrates together in a proper oirentation.
A molecule other than water plays the role of a proton donor or acceptor.
Metal ion catalysis
–
Metal ions can serve as electrophilic catalyst, stabilizing negative charge on a reaction
intermediate.
Catalytic Strategies
Approximation
Enzyme serves as a template to bind the substrates so that they are close to each
other in the reaction center.
- Bring substrate into contact with catalytic groups or other substrates.
- Correct orientation for bond formation.
- Freeze translational and rotational motion.
Catalytic Strategies
Approximation
a)
Bimolecular reaction (high
activation energy, low rate).
b)
Unimolecular reaction, rate
enhanced by factor of 105 due to
increased probability of
collision/reaction of the 2 groups
c)
Constraint of structure to orient
groups better (elimination of
freedom of rotation around bonds
between reactive groups), rate
enhanced by another factor of 103,
for 108 total rate enhancement
over bimolecular reaction
Catalytic Strategies
•
Covalent catalysis
The principle advantage of using an active site residue instead of water
directly is that formation of covalent linkage leads to unimolecular reaction,
which is entropically favored over the bimolecular reaction.
Enzyme that utilize covalent catalysis are generally two step process:
formation and breakdown of covalent intermediate rather than catalysis of
the single reaction directly.
 Y should be a better leaving group than X.
 X is a better attacking group then Z.
 Covalent intermediate should be more reactive than substrate.
Catalytic Strategies
•
Covalent catalysis
ATP-Dependent DNA Ligase
Lys
Lys
NH2
N
N
N
LigaseﾐAdenylate
H2N
H2N
O
H2C
O
O
O
OH
OH
O
N
O
O
P
ATP
NH
P
P
N
N
N
O
N
O
O
H2C
O
+
O
OH
P
OH
O
O
P
P
O
O
H
Phosphoramidate
Intermediate
O
O
Lys
+
O
N
P
H
O
O
Nucleoside
O
O
O
O
Catalytic Strategies
•
Covalent catalysis
What kind of groups in proteins are good nucleophiles:
•
•
•
Aspartate
caboxylates
Glutamates
caboxylates
Cystine
thiol-
Serine
hydroxyl-
Tyrosine
hydroxyl-
Lysine
amino-
Histadine
imidazolyl-
Catalytic Strategies
Acid-base catalysis
A proton (H+) is transferred in the transition state.
Specific acid-base catalysis:
Protons from hydronium ion (H3O+) and hydroxide ions (OH-) act directly
as the acid and base group.
General acid-base catalysis:
• Catalytic group participates in proton transfer stabilize the transition state
of the chemical reaction.
• Protons from amino acid side chains, cofactors, organic substrates act as
Bronsted-Lowry acid and base group.
Catalytic Strategies
Acid-base catalysis
Transition State of Stabilization by a General Acid (A) or General Base (B) in Ester
Hydrolysis by Water.
Transition state can be stabilized by
acid group (A-H) acting as a partial
proton donor for carbonyl oxygen of the
ester - Enhance the stability of partial
negative charge on the ester.
Alternatively, enzyme can stabilize
transition state by basic group (B:)
acting as proton acceptor.
For even greater catalysis, enzyme can
utilize acid and base simultaneously
Catalytic Strategies
Acid-base catalysis
Histidine pKa is around 7. It is the most effective general acid or base.
Example: RNase A:
 His 12
 General Base
 Abstracts a proton from 2’ hydroxyl of
3’ nucleotide.
 His 119
 General acid
 Donates a proton to 5’ hydroxyl of
nucleoside.
Catalytic Strategies
Acid-base catalysis
Histidine pKa is around 7. It is the most effective general acid or base.
Example: RNase A:
 His 12
 General Base
 Abstracts a proton from 2’ hydroxyl of
3’ nucleotide.
 His 119
 General acid
 Donates a proton to 5’ hydroxyl of
nucleoside.
2’-3’ cyclic phosphate intermediate
Net Proton Transfer from His119 to His12
Catalytic Strategies
Acid-base catalysis
Histidine pKa is around 7. It is the most effective general acid or base.
Example: RNase A:
 His 12
 General Base
 Abstracts a proton from 2’ hydroxyl of
3’ nucleotide.
 His 119
 General acid
 Donates a proton to 5’ hydroxyl of
nucleoside.
Water replaces the released nucleoside
Acid and base roles are reversed for H12 and H119
Catalytic Strategies
Acid-base catalysis
Histidine pKa is around 7. It is the most effective general acid or base.
Example: RNase A:
 His 12
 General Base
 Abstracts a proton from 2’ hydroxyl of
3’ nucleotide.
 His 119
 General acid
 Donates a proton to 5’ hydroxyl of
nucleoside.
Original Histidine protonation states are restored
General Acids
COOH
CH2 OH
CH2
OH
NH3
SH
CH2
HN
NH
General Bases
NH2
COO
S
CH2
HN
N:
Catalytic Strategies
Metal ion catalysis.
Metal ions can …
•
Electrostatically stabilizing or shielding negative charges.
•
Act to bridge a substrate and nucleophilic group.
•
Bind to substrates to insure proper orientation.
•
Participate in oxidation/reduction mechanisms through change of oxidation
state.
Catalytic Strategies
Metal ion catalysis.
1)
Can stabilize developing negative charge on
a leaving group, making it a better leaving
group.
Catalytic Strategies
Metal ion catalysis.
1)
Can stabilize developing negative charge on
a leaving group, making it a better leaving
group.
2)
Can shield negative charges on substrate
group that will otherwise repel attack of
nucleophile.
Catalytic Strategies
Metal ion catalysis.
1)
Can stabilize developing negative charge on
a leaving group, making it a better leaving
group.
2)
Can shield negative charges on substrate
group that will otherwise repaile attack of
nucleophile.
3)
Can increase the rate of a hydrolysis
reaction by forming a complex with water,
thereby increasing water’s acidity.
Specific Forces Involved in Enzyme-Substrate
Complex Formation
• 1. Covalent Bond
• 2. Ionic (or Electrostatic) Interactions
• 3. Ion-Dipole and Dipole-Dipole
Interactions
• 4. Hydrogen Bonds
• 5. Charge Transfer Complexes
• 6. Hydrophobic Interactions
• 7. Vander Waals Forces