Biomedical Engineering Reference
In-Depth Information
state, the resulting positive or negative charge makes the transition state unfavorable. The
presence of acids or bases results in a stabilization of such a transition state. By providing
acid or base groups at the active site, an enzyme is thus able to stabilize the charged transition
state. This mechanism is employed by a wide variety of enzymes.
Two types of acid or base catalysis can be distinguished: general and specific. The distinc-
tion between specific and general acids and bases can be best understood by examining
experimental observations of catalytic reaction rates. Consider, for example, the hydrolysis
of an ester in buffered solutions. The hydrolysis rate can be determined at a constant pH
(by maintaining the ratio of acid and base forms of the buffer at a constant value), at several
different total concentrations of buffer. If the rate of reaction increases with increasing buffer
concentration, then the buffer must be involved in the reaction and act as a catalyst. This is
general acid or base catalysis. If the rate is unaffected by buffer concentration, then the reac-
tion involves specific acid or base catalysis. The reacting species would be only proton (H
รพ
)or
hydroxyl anion (OH
), and the buffer simply serves to maintain these species at constant
concentrations.
How does an enzyme obtain the rate enhancements made possible by general acid
e
base
catalysis? The answer lies in the combination of pK
a
values for amino acid moieties involved
in acid
e
base catalysis and the typical values of proton dissociation constant K
a
, describing
proton transfer. An acid with a pK
a
of 5 is a better general acid catalyst than one with
apK
a
of 7. At pH 7, however, typical of the optimal pH of many enzymes, only 1% of the
acid with a pK
a
of 5 will be unionized and active in catalysis. The acid with a pK
a
of 7 will
be 50% unprotonated. The same trend applies to base catalysis. If we consider the amino
acids, we see that histidine contains an imidazole moiety which has a pK
a
value around
6
e
7. Therefore, histidine is widely found in enzymes involved in base catalysis as it is
50% ionized at neutral pH. The imidazole moiety may thus be considered as the most effec-
tive amino acid base existing at neutral pH. In fact, all enzymatic acyl transfers catalyzed by
proteases (e.g. trypsin, chymotrypsin) involve histidine. The imidazole group of histidine can
also function as a nucleophile, and we must be careful to determine whether histidine acts
a general base catalyst or a nucleophilic catalyst. In proteases, histidine functions as a base
catalyst, but it typically found closely associated with serine. Histidine is thought to deprot-
onate this neighboring serine alcohol moiety by general base catalysis. The serine alkoxide
ion (
O
e
CH
2
CH
e
Enz) so generated has a pK
a
of 13.7 and is thus a stronger base than histi-
dine but at neutral pH is less reactive. Serine functions primarily as a strong nucleophile in
proteases.
In addition to covalent and general acid
e
base catalysis, enzymes also employ other
mechanisms of rate enhancement. One of these is electrostatic catalysis. As we have seen
from the transition state analysis described above, electrostatic interactions between
substrate and enzyme may stabilize this transition state and thus yield significant rate
enhancements. We shall briefly describe this and the other types of catalysis that are found
in enzymes.
Electrostatic Catalysis. In water, the large dielectric constant results in a small electro-
static interaction energy between charges, and electrostatic catalysis is not generally impor-
tant in homogeneous catalysis in aqueous systems. However, the active site of a protein is
very heterogeneous, and the dielectric constant of the medium between charged groups
may be quite different from water. The aromatic and aliphatic amino acid residues present
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