STRUCTURAL AND FUNCTIONAL ROLES OF CYSTEINE-90 AND CYSTEINE-240 IN S-ADENOSYLMETHIONINE SYNTHETASE

Site-specific mutagenesis was performed on the structural gene for Escherichia coli S-adenosyhnethionine (AdoMet) synthetase to introduce mutations at cysteines 90 and 240, residues previously implicated by chemical modification studies to be catalytically and/or structurally important, The AdoMet synthetase mutants (i.e, MetK/C90A, MetK/C90S, and MetK/C240A) retained up to similar to 10% of wild type activity, demonstrating that neither sulfhydryl is required for catalytic activity. Mutations at Cys-90 produced a mixture of noninterconverting dimeric and tetrameric proteins, suggesting a structural significance for Cys-90, Dimeric Cys 90 mutants retained similar to 1% of wild type activity, indicating a structural influence on enzyme activity. Both dimeric and tetrameric MetK/C90A had up to a similar to 70-fold increase in K-m for ATP, while both dimeric and tetrameric MetK/C90S had K-m values for ATP similar to the wild type enzyme, suggesting a linkage between Cys-90 and the ATP binding site, MetK/C240A was isolated solely as a tetramer and differed from wild type enzyme only in its 10-fold reduction in specific activity, suggesting that the mutation affects the rate-limiting step of the reaction, which for the wild type enzyme is the joining of ATP and L-methionine to yield AdoMet and tripolyphosphate. Remarkably all of the mutants are much more thermally stable than the wild type enzyme.