Site-directed mutagenesis of rat liver S-adenosylmethionine synthetase. Identification of a cysteine residue critical for the oligomeric state.

We have examined the functional importance of the cysteine residues of rat liver S-adenosylmethionine synthetase. For this purpose the ten cysteine residues of the molecule were changed to serines by site-directed mutagenesis. Ten recombinant enzyme mutants were obtained by using a bacterial expression system. The same level of expression was obtained for the wild type and mutants, but the ratio of S-adenosylmethionine synthetase between soluble and insoluble fractions differed for some of the mutant forms. The immunoreactivity against an anti-(rat liver S-adenosylmethionine synthetase) antibody was equivalent in all the cases. Effects on S-adenosylmethionine synthetase activities were also measured. Mutants C57S, C69S, C105S and C121S showed decreased relative specific activity of 68, 85, 63 and 29%, respectively, compared with wild-type, whereas C312S resulted in an increase of 1.6-fold. Separation of tetramer and dimer forms for wild type and mutants was carried out by using phenyl-Sepharose columns. The dimer/tetramer ratio was calculated based on the activity and on the protein level estimated by immunoblotting. No monomeric forms of the enzyme were detected in any case. Comparison of dimer/tetramer ratios indicates the importance of cysteine-69 (dimer/tetramer protein ratio of 88 versus 10.2 in the wild type) in maintaining the oligomeric state of rat liver S-adenosylmethionine synthetase. Moreover, all the mutations carried out of cysteine residues between cysteine-35 and cysteine-105 altered the ratio between oligomeric forms.