Site-specific mutagenesis was performed on the structural gene for Escherichia coli S-adenosylmethionine (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 approximately 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 approximately 1% of wild type activity, indicating a structural influence on enzyme activity. Both dimeric and tetrameric MetK/C90A had up to a approximately 70-fold increase in Km for ATP, while both dimeric and tetrameric MetK/C90S had Km 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.