Chemical Enzyme Modifications

Covalent chemical modification of enzymes can be regarded as the original method available for altering enzyme properties. This long history does not, however, mean that this kind of basic enzyme engineering has lost anything of its attraction, chemical modification has re-emerged as a powerful complementary technique to genetic approaches for tailoring enzymes. Several benefits contribute to this renaissance:

1. chemical modification is generally applicable;

2. it is usually inexpensive and easy to perform; and

3. it enables incorporation of non-coded amino acid moieties and, thus, leads to a variety of enzyme species which cannot be generated by routine genetic engineering.

Classical approaches to chemical enzyme modification, however, often suffer from lack of regio-selectivity, which can yield heterogeneous and irreproducible enzyme mixtures. For example, preparation of methyl-chymotrypsin, -subtilisin or -trypsin using methyl sulfonate reagents, originally used to methylate the histidine of the catalytic triad of the enzymes, yields enzyme mixtures in which the remaining histidines of the enzyme molecules are partly or completely methylated [2]. Because of the small size of the modifying moiety, however, undesired effects of this random modification on enzyme activity are only marginal. The methylated enzyme variants are now recognized as interesting biocatalysts for peptide synthesis, mainly because of loss of proteolytic activity with some of the esterase activity remaining. Although important, the very low synthetic activity of methylated proteases should, however, generally hinder their practical use. Oxidation of Met192 in chymotrypsin leads to a much more active enzyme variant which, however, has still proteolytic activity. Nevertheless, because of the improved esterase to amidase ratio and the higher stability of the enzyme towards basic conditions, the Met192-sulfoxide-chymotrypsin was found to be a useful enzyme for peptide synthesis. The preparation of seleno-subtilisin and thiol-subtilisin can be regarded as further examples of small-size chemical modification [14]. The latter, which simultaneously marks the beginning of chemical enzyme engineering, entails conversion of the active-site serine 221 of subtilisin to cysteine. This first alteration remains one of the most useful. Similar to methylated proteases, subtilisin S221C is catalytically wounded to the point that it will barely hydrolyze peptide bonds yet is quite reactive with certain activated ester substrates. Thiol-subtilisin also benefits from the fact that thiol esters usually have higher aminolysis to hydrolysis ratios than regular oxo esters. This combination of properties has made it a useful tool for peptide synthesis and transesterification reactions [2]. It should be noted, however, that its catalytic activity is several orders of magnitude lower than that of the wild-type enzyme, although it is more active than the His-methylated species. Seleno-subtilisin is a much poorer enzyme than thiol-subtilisin. Its synthetic application for peptide synthesis essentially needs the use of highly activated esters as the acyl donors, but even then the reactions proceed very slowly. In addition, the enzyme is very sensitive to oxidants. Although less suitable for peptide synthesis, this behavior has made seleno-subtilisin a useful biocatalyst for mediation of peroxidase-like reactions [15].

Site-specific modifications essentially need the presence of a unique amino acid moiety which can undergo chemo-selective reactions. Having such a unique amino acid within an enzyme is, however, the exception rather than the rule. Subtilisin and carboxypeptidase Y can be regarded as such rare enzymes, because they contain no natural cysteines. Thus, incorporation of an artificial cysteine moiety by site-directed mutagenesis creates a unique reaction center that can be chemically modified to introduce an unnatural amino acid side-chain selectively (Scheme 5.1.6) [16]. By use of subtilisin a variety of structures differing in size and phys-icochemical properties have been covalently linked to artificial cysteines located at different positions within the enzyme. Interestingly, remarkable effects on both catalytic properties were observed. Although most chemical active-site modifications reduce the activity of an enzyme this was not always so for this type of active-site engineering. For example, incorporation of hydrophobic ligands at the enzyme's S2 subsite increased the activity of the enzyme more than threefold compared with the parent enzyme [16c]. An improved ratio of esterase to amidase activity site-directed mutagenesis thiol-selective SH modification

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