engleski

SPECIFICITY MODULATION IN FUNCTIONAL ACTIVE SITE MUTANTS OF YEAST SERYL-tRNA SYNTHETASE

Seryl-tRNA synthetase is a class II aminoacyl-tRNA synthetase for which we previously demonstrated the tRNA-dependent amino acid recognition. In this work we constructed two new mutants in motif 2 loop, the part of active site which is involved in binding of ATP, amino acid and the acceptor end of tRNA. One mutant containing three amino acid substitutions (K287T/D288Y/A289V) exhibited the impaired tRNA-dependent recognition, while the other mutant containing only single conservative substitution (K287R) showed only mild increase in amino acid affinity. In our kinetic experiments we observed that serine-hydroxamate, rather strong competetive inhibitor of E.coli SerRS, is surprisingly good supstrate for the yeast enzyme, with Km an order of magnitude higher compared to Km for serine. The kcat was comparable to kcat of reaction with serine. When serine-hydroxamate was used as a substrate in the reaction of activation (PPi-exchange), kcat was less impaired for the mutant with three substitutions (K287T/D288Y/A289V), than for wild-type enzyme. We conclude that alternations in active site relax serine-hydroxamate discrimination.

tRNA:seryl-tRNA synthetase recognition; substrate analogues

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