Enzymes - Bioprocess Engineering: Kinetics, Biosystems, Sustainability and Reactor Design - page 333

at the active site act to reduce the dielectric constant and charged amino acid residues act as
fixed dipoles, thus stabilizing charge quite effectively. The electrostatic interaction energy
depends on the charges and is inversely proportional to the dielectric constant and distance
between charges.
Lowering the dielectric constant can increase this energy considerably. Proteins may thus
use parts of their own structure to solvate transition states and induce electrostatic strain. In
fact, enzymes may stabilize charged groups in the transition state better than water, as the
amino acids which function as dipoles are rigidly positioned and have a direction in relation
to the substrate, whereas in water this directionality is lost.
The overall significance of electrostatic catalysis in enzymes is still not clear, as determina-
tion of the local dielectric constant is difficult. Electrostatic stabilization of charged transition
states certainly plays some role, however.
Catalysis Involving Metal Ions. In metalloenzymes, a metal ion is present at the active
site, and this ion plays an important role in stabilizing negative charges that are formed in
electrophilic catalysis. Zinc, copper, and cobalt are commonly involved in coordination of
oxyanions involved as reaction intermediates. The enzyme carboxypeptidase-A, which is
a carboxyl-terminus exopeptidase (i.e. it acts by hydrolyzing the peptide from the carbox-
ylic acid terminus), contains Zn 2 þ which polarizes the carbonyl oxygen of the terminal
peptide bond. The terminal carboxylate is charge paired with the guanidinium cation of
Arg 145 leading to polarization of the terminal carboxylic carbonyl group. This polariza-
tion increases the electrophilicity of the carbonyl carbon and facilitates nucleophile-
mediated hydrolysis of the amide bond. This is illustrated in Fig. 8.6 . In addition to
stabilizing negative charges, metal ions serve as a source of potent nucleophilic hydroxyl
ions. Metal-bound water molecules provide these nucleophilic hydroxyl groups at
neutral pH.
An example is the extremely rapid hydration of CO 2 by carbonic anhydrase to produce
bicarbonate. The enzyme contains zinc coordinated to the imidazole groups of three histi-
dines, with the fourth ligand being water, which is ionized. The zinc-bound hydroxyl has
apK a of 8.7, and the reaction mechanism is thought to that shown in Fig. 8.7 .
Also, enzymes may hold the substrates at certain positions and angles to improve the reac-
tion rate, which is known as the orientation effect. In some enzymes, the formation of an ES

Cleavage

H

R

COO _

+

R '

N Arg 145

C C

H

O

Zn 2+

FIGURE 8.6 Metalloenzyme carboxypeptidase-A facilitates nucleophile-mediated hydrolysis of an amide bond.