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Enzyme Catalysis

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.

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.

 Here we have the formation of an anhydride from the

reaction of an ester with a carboxylate ion.
If we covalently link the two reacting groups together,
than the close proximity to each other increases the
effective concentration and we get a 105 fold enhancement
of the rate.
The covalently linked molecule still has a lot of rotational
freedom. The rotational freedom was been removed in the
last molecule shown here. The cyclic molecule with its
bridge and double bond has little rotational freedom and
therefore enhanced rate of anhydride formation.
In addition to proximity, enzymes also bind substrates in the optimal orientation for reacting. Enzymes
bind substrates in a manner that immobilizes them and aligns them for maximal reactivity. The free
energy required to do so is derived from the specific binding energy of substrate to enzyme.

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.

II. Acid-base catalysis

Specific Acid-base catalysis is when the H+ or OH ion catalyzes the reaction. In this case, the rate of
catalysis is proportional to the pH.
General Acid catalysis is a process of partial proton transfer from a weak acid to the substrate which
lowers the free energy of the reactions transition state. In this case at a given pH, the rate of catalysis is
proportional to the concentration of the weak acid.
General Base catalysis is the process of partial proton abstraction by a weak base from the substrate
which lowers the activation energy. In this case at a given pH, the rate of catalysis is proportional to the
concentration of the weak base.

This is an example of specific acid-base catalysis. The buffer is our

weak acid or weak base. The catalytic rate is independent of the
concentration of the buffer, it solely depends on the pH.

This is an example of general acid-base catalysis. At a given pH,

increasing the weak acid or weak base concentration increases the
rate of the reaction.
Keto-enol Tautorization

Mechanism of keto-enol tautomerization.

(a) Uncatalyzed, the development of a
carbanion in the transition produces a high
activation energy resulting in slow rate.
(b) General acid catalysis, the partial
donation of the proton by the weak acid
reduces the carbanion character of the
transition state and lowers the activation
(c) General base catalysis, the partial
abstraction of the methyl proton by the
general base gives the base a partial positive
charge which helps delocalize the negative charge of the transition state and lowers the activation energy.
Enzymes often catalyzed concerted general acid-base catalysis.








+ H+

B: + H+

In order for an enzyme to catalyze a reaction by general base

catalysis, the active site residue that functions as the general
base has to be deprotonated. At a pH below the pKa of the
general base, the side chain is protonated and can no longer
function as general base.

The opposite is true for an enzyme that relies on general

acid catalysis. The active site residue that functions as the
general acid has to be protonated, so at a pH above the pKa
of the general acid, the side chain becomes unprotonated
and can no longer function as a general acid.

Many enzymes employ both general acid and general base

catalysis. In this case, there is general base that has to be
unprotonated to have catalytic activity, and there is a general
acid that has to protonated for full activity. The enzymes
maximal activity will occur at pH range above the pKa of
the general base and below the pKa of the general acid.
Example of Acid-Base catalysis- RNAase A.
RNAase is a digestive enzyme produced in the pancreas and secreted into the small intestine where it
digests RNA molecules to their component nucleotides.

The RNAase enzyme catalyzes the hydrolysis of RNA molecules

by concerted general acid-base catalysis. The first step on
enzyme catalysis is the formation of the enzyme-RNA complex.
Once the substrate RNA is bound, His-12 functions as a general
base partially abstracting a hydrogen from the 2-hydroxyl of the
near by ribose. This promotes nucleophilic attack of the 2hydroxyl group at the phosphorous center. His-119 concertedly
acts as general acid partially donating a proton to the leaving

A 2,3 cyclic intermediate is formed which can be isolated. The

second step of the RNAase catalyzed reaction is essentially the
reverse of the first step. A water is activated for nucleophilic
attack by general base catalysis of His-119 with His-12
functioning as a general acid donating its hydrogen to the 2hydroxyl group.

The last step is the dissociation of the RNA molecule from the
active site leaving the enzyme ready for another round of

III Covalent Catalysis

Many enzymes accelerate reaction rates through the formation of covalent bonds between the enzyme and
the substrate. A common reaction is group transfer reaction shown below.
BX + Y

BY + X



B +Y


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.













Acyl enzyme intermediate










Phosphoryl enzyme













Glucosyl enzyme intermediate

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.





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.

The uncatalyzed reaction is shown on the top.

IV Metal Ion Catalysis
Many enzymes require metal cofactors for maximal activity. If the enzyme binds the cofactor metal very
tightly it is called a metalloenzyme. If the enzyme binds the metal weakly, then it is called a metal
activated enzyme.
Metals can function in a lot of roles. Metals often act as electrophilic catalysts, stabilizing the formation
of negative charges in the transition state that often develop during reactions. Metals often coordinate a
water molecule and increase the acidity of water to generate nucleophilic hydroxides at the physiological
pH. Because metals can exist in multiple oxidation states, they often function in oxidation or reduction
reactions. Metal can also coordinate substrate molecules to orient them for optimal catalysis.
An example of a metal functioning as an electrophilic catalyst is the Cu2+ or
Ni2+ catalyzed decarboxylation of dimethylocaloacetate.

Here the metal ion is denoted by Mn+ chelates to two oxygens of

dimethyloxaloacetate which stabilizes the developing negative charge of the
transition state and facilitate the decarboxylation reaction.

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
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

Changing the pH alters the rate of carbon dioxide hydration by

carbonic anhydrase. The rate of activity is dependent on the
protonation state of the coordinated water molecule.
The zinc complex increases the acidity of the coordinated water
molecule such that the pKa of the water molecule has been
shifted from 14 to 7.

Mechanism of carbonic anhydrase:

1 The coordination of a molecule of water to the zinc
complex facilitates the generation of the hydroxide ion.
2 Carbon dioxide binds to the active site.
3 The hydroxide ion attacks the carbon dioxide producing
the bicarbonate ion.
4 The catalytic site is regenerated by the release of
bicarbonate and the coordination of another molecule of

Histidine 64 accelerates the reaction by

abstracting the proton from the zinc coordinated water molecule.