For example, although the overall reaction catalyzed by
acetylcholinesterase consists of hydrolysis of acetyl-
choline to choline and acetic acid, the detailed mechanism
is a two-step displacement reaction in which an alcohol
(choline) is produced first, followed by an acid (acetic
acid). The same is true for the rhodanese-catalyzed reac-
tion. The types of experiments that are carried out to arrive
at the description of an enzyme mechanism are as follows.
Isotope Exchange
If a particular atom of the substrate
or product is labeled with an isotope other than the natural
one, its location at various intermediate reaction steps can
be followed, providing information on what is happening
to that part of the molecule.
Irreversible Inhibition Studies
If an enzyme is inac-
tivated by a reagent that reacts with a specific amino acid
residue, then one (or more) of the reagent-specific amino
acid residues is involved in catalysis by that enzyme.
pH Dependence
Usually only one form (protonated
or unprotonated) of an acidic or basic residue is involved
in a catalysis.
Structural studies, particularly those that elucidate the
three-dimensional relationships of the amino acid residues
in the enzyme, permit judgments about whether the in-
volvement of one or the other side chain is physically and
sterically possible. Such techniques include nuclear mag-
netic resonance, electron spin resonance, single-crystal
x-ray diffraction, and cross-linking studies. The impor-
tant question to be answered by mechanistic studies is
how an enzyme catalyzes a particular reaction so rapidly.
Two general factors seem to be involved.
1. Because all the reactants are bound to a small region
on the enzyme, their effective concentrations are
greatly increased; i.e., the adjacent binding makes it
easier for an effective collision to occur between
2. As a reaction proceeds, the conformation of the
reacting molecules must change from that of the
substrates to that of the products. Alterations in
bonds, bond lengths, and bond angles are involved.
The activation energy represents, at least in part, the
work needed to bring about this transformation. The
conformation that lies between those of the substrates
and products and that has the highest energy is termed
transition state.
Additionally, the binding energy
of an enzyme distorts the substrates, so that they
approach or attain the conformation of the transition
state. Detailed structures and catalytic mechanisms
are known for many enzymes, some of which are
discussed elsewhere (e.g., lysozyme in Chapter 11).
Serine proteinases are among the most thoroughly
studied enzymes, and they share similar catalytic
mechanisms (see above). However, they act on
different substrates because of differences in their
substrate binding sites. Chymotrypsin, trypsin, and
elastase have the same amino acid sequence around
the active serine residue (*),
and their tertiary structures, as revealed by x-ray
studies, are similar. Their amino acid sequences are
40% identical, leading to the hypothesis that these
three enzymes may have evolved from a common
ancestral gene. Duplication of this gene probably
occurred several times during evolution and, with
mutation, eventually yielded genes that now code for
these three proteinases, each with its own substrate
specificity. This type of evolution is known as
divergent evolution.
Chymotrypsin and subtilisin
provide an example of
independent convergent
Subtilisin is a serine proteinase of bacterial
origin whose mechanism of action is similar to that of
Chymotrypsin, but it differs from Chymotrypsin in the
amino acid sequence around the active serine residue:
Chymotrypsin and subtilisin also differ in their amino
acid sequences, number of disulfide bridges
(chymotrypsin has five, whereas subtilisin has none),
and overall three-dimensional structures. The striking
difference in structure and common catalytic
mechanism are taken as evidence of an independent
but convergent evolutionary process.
Coenzymes, Prosthetic Groups, and Cofactors
Many enzymes require the presence of low-molecular-
weight nonprotein molecules. These small molecules may
be bound to the enzyme by a covalent, tight, or noncovalent
linkage. Prosthetic groups are usually bound by covalent
or tight linkages and coenzymes by noncovalent linkages.
Cofactors usually are metal ions or organic compounds
(some are vitamins, e.g., ascorbic acid).
Prosthetic groups and coenzymes are complex organic
compounds, many of which are derived from vitamins.
These compounds are recycled and are needed only in
catalytic amounts to convert a large amount of reac-
tants to products. Coenzymes function as substrates in
Enzymes I: General Properties, Kinetics, and Inhibition
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