Download General acid-base catalysis

Advanced Bioprocess Engineering
Enzymes & Enzymes Kinetics
Lecturer Dr. Kamal E. M. Elkahlout
Assistant Prof. of Biotechnology
Enzyme (Basic Principle)
III. Other 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).
Enzyme (Basic Principle)
III. Other 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
Rate:
108
Acylic phospodiester
1
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.
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
•
Covalent catalysis
Schiff Base Formation
•
A Schiff base may form from the condensation of an amine with a carbonyl
compound.
•
The Schiff base (protonated at neutral pH) acts as an electron sink that
greatly stabilizes negative charge that develops on the adjacent carbon.
Stable Intermediate
Catalytic Strategies
•
Covalent catalysis
Schiff Base Formation
•
Enzymes that form Schiff base intermediates are typically irreversibly
inhibited by the addition of sodium borohydride (Na+ BH4–).
•
Borohydride reduces the Schiff base and “traps” the intermediate such that it
can no longer be hydrolyzed to release the product from the enzyme.
•
This is often used as evidence for a mechanism involving an enzyme-linked
Schiff base intermediate.
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 protein 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
Catalytic Strategies
Metal ion catalysis.
Metal ions can …
•
Electrostatically stabilizing or shielding negative charges.
•
Act much like a proton but can be present in high concentration at neutral pH
and can have multiple positive 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.
Metal ions can …
•
Electrostatically stabilizing or shielding negative charges.
•
Act much like a proton but can be present in high concentration at neutral pH
and can have multiple positive 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.
Catalytic Strategies
Metal ion catalysis.
Examples: