nucleophilic amino acid with an unreactive one on the catalytic properties of the
enzyme, conclusions can be drawn about the role of the amino acid residue that has
been replaced. In principle it is also possible to produce variants that are more active
than the native enzyme. Such studies are based on knowledge of the protein structure,
function and mechanism of course and assume that the impact of the single amino
acid replacement is confined to the active site and has not affected other aspects of the
enzyme’s structure. This needs to be confirmed by complementary structural studies,
for example, spectroscopic techniques. Thisrational redesignapproach has resulted in
the generation of a superoxide dismutase with an enhanced activity relative to the
native enzyme and an isocitrate dehydrogenase with specificity different to that of the
native form.15.4.2 Catalytic mechanisms
The application of the various strategies outlined above to a wide range of enzymes has
enabled mechanisms to be deduced for many of them. Crystallographic and site-directed
mutagenesis studies have been particularly successful in providing detailed information
about the stereochemical and electronic events involved in substrate binding and product
formation. The most commonly occurring amino acid residues in enzyme catalytic sites
are the imidazole group of histidine, the guanidinium group of arginine, the carboxylate
groups of glutamate and aspartate, the amino group of lysine, the hydroxyl groups of
serine, threonine and tyrosine and the thiol group of cysteine. Of these nine groups, the
imidazole group of histidine appears to be quantitatively the most important. These
specific amino acid side chains commonly occur in pairs or triplets within catalytic sites.
In the lipases and peptidases thecatalytic triadserine, aspartate and histidine occurs very
commonly. In chymotrypsin for example, the three amino acids are located in positions
195, 102 and 95 respectively and are brought into juxtaposition by the three-dimensional
folding of the protein chain. Their roles include:- to activate the substrate by forming a hydrogen bond thereby lowering the reaction
activation energy barrier. Such a hydrogen bond has been referred to as alow-barrier
hydrogen bond; - to provide a nucleophile to attack the substrate;
- to provide the components for the acid–base catalysis of the substrate of the type well
known in conventional organic chemistry; - to stabilise the transition state of the reaction.
The quantitative importance of histidine can be explained by the facts that it is the
only residue that has a pKanear neutral so that it can easily function as an acid–base
catalyst, and that it can also easily function as a nucleophile and use its charged form to
stabilise transition states. Whilst these amino acid side chains provide the components
for catalysis, the specificity of the reaction is determined by the three-dimensional
structure of the enzyme and the microenvironment it creates within the active site.
The experimental techniques discussed above have successfully identified key
amino acids involved in the catalytic process. However, the question remains as to
the nature of the factors limiting the rate of catalysis. Recent studies, particularly614 Enzymes