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Bryant Miles
Previously, we learned that enzymes stabilize the transition state by tightly binding to it. When the
enzyme binds the substrate to form the enzyme-substrate complex the binding energy is used to lower the
activation energy and help drive catalysis.
Proximity
Enzymes have specific binding sites for their substrates. When an enzyme binds a substrate, it essentially
removes the substrate from the dilute solution and holds it in close proximity to the reactive functional
groups of the enzyme, another substrate or a cofactor. The close proximity raises the effective
concentration much greater than the concentration of the substrate in the solution. This increase in
effective concentration leads to an increased reaction rate.
Catalytic Mechanisms
Three Types of Catalytic Mechanisms
1.) Acid-base catalysis
2.) Covalent catalysis
3.) Metal ion catalysis
4.) Electrostatic catalysis
I. Electrostatic catalyst: The binding of substrate generally excludes water from the enzymes active site.
The dielectric constant of proteins hydrophobic interiors is low, similar to organic solvents. Thus,
electrostatic interactions are stronger than in the aqueous solution. The low dielectric constant can greatly
enhance chemical reactivity. Charge distributions at the active site are arranged to stabilize the charges
developed in formation of the transition state.
R
R
C
R
O
CH2
B:
C
O
CH2
B
A-
CH2
+
+ H+
B: + H+
The last step is the dissociation of the RNA molecule from the
active site leaving the enzyme ready for another round of
catalysis.
BY + X
X + ENZ
ENZ
B +Y
ENZ + B
Typically the covalent enzyme-substrate bond is formed by a nucleophilic substitution reaction where a
nucleophile of the enzyme attacks an electrophilic center of the substrate. This is also called nucleophilic
catalysis. In order to catalyze the group transfer reaction shown above, the enzymes functional group
serving as the nucleophile has to a better nucleophile than Y and conversely the same functional group
has to be a better leaving group than X.
The side chains of amino acids found in proteins offers a variety of nucleophilic centers which includes
amines (K,R), carboxylates (D,E), hydroxyls (S,T & Y), imidazoles (H) and thiols (S). All of these
groups can function as a nucleophile to attack electrophilic centers. Typical electrophilic centers of
substrates include phosphoryl groups, acyl groups and glycosyl groups. The reaction of an enzyme
nucleophile and the substrate electrophilic center results in covalently bonded enxyme-substrate
intermediate. Examples are shown below.
O
O
R
R'
O
-
OENZ
O
O
R'
Y
ENZ
P
O
O
Y
NU
ENZ
NU
NU
HO
HO
HO
HO
ENZ
NU
H
H
ENZ
NU
-
Phosphoryl enzyme
intermediate
R'O-
O-
NU
ENZ
NU
ENZ
OH
NU
ENZ
OH
H
OH
OH
The covalent enzyme intermediates formed are in turn attacked by a nucleophilic second substrate (often
water) producing the desired product.
Another example of covalent catalysis are enzymes that form Schiff bases through the nucleophilic attack
of an amine (typically lysine) on a carbonyl shown below.
H
:
O
OH
:B
-
OH
In Schiff base formation, a nucleophilic amine attacks the carbonyl group to form an imine bond called a
Schiff base. The protonated imine of this covalent intermediate acts as an electron sink stabilizing
transition states with high energy carbanion character. This mechanism of covalent catalysis involves
three steps (1) the nucleophilic attack of the amine on the carbonyl, (2) The electrophilic withdraw of
electrons by the protonate imine which is an example of electrophilic catalysis. (3) The nucleophilic
attack of water to eliminate the amine. An example of this is the catalysis of the decarboxylation of
acetoacetate by a primary amine.
Carbonic Anhydrase
An example of a metal ions charge lowering the pKa of a coordinated water molecule is carbonic
anhydrase which is found in high concentrations in erythrocytes, to catalyze the conversion of CO2 to
bicarbonate.
CO2
HCO3- + H+
The active site of carbonic anhydrase is
shown. The active site is found in deep
cleft where a zinc molecule is coordinated
to three histidine residues and a water
molecule. Zn2+ polarizes water,
increasing its acidity. This results in
having a Zn bound hydroxide ion which
attacks a bound CO2 molecule to generate
bicarbonate.