STRUCTURAL AND FUNCTIONAL ROLES OF THE CYSTEINE RESIDUES OF BACILLUS-STEAROTHERMOPHILUS FARNESYL DIPHOSPHATE SYNTHASE

p-(Chloromercuri)benzoic acid inhibited the catalytic activity of Baci llus stearothermophilus farnesyl diphosphate synthase (FPP synthase), which contains only two cysteine residues at positions 73 and 289. In order to explore the role of the cysteine residues, either or both of them were replaced with phenylalanine or serine. Five mutant enzymes, C73F, C73S, C289F, C289S, and C73S-C289S, were overproduced in Escheri chia coli and purified to homogeneity. All of them were active as farn esyl diphosphate synthase, showing specific activities comparable to t hat of the wild-type enzyme. These results indicate that neither of th e cysteines is essential for catalytic function. The C73F mutant, howe ver, was very sensitive to heat treatment, while C73S was as highly st able as the wild type. The K-m value of C289F for isopentenyl diphosph ate is 10 times that of the wild type. The wild-type enzyme was conver ted into an oxidized form which was separable from the native enzyme o n ion-exchange chromatography, and this conversion was accelerated by cupric ions. Similar conversion has previously been reported by severa l researchers, who found the occurrence of two forms of pig liver FPP synthase and who attributed this phenomenon to the oxidoreduction of s ulfhydryl and disulfide groups. However, even the C73S-C289S mutant, w hich has no cysteine residues, was also converted into an oxidized for m as in the case of the wild-type enzyme. These results provide eviden ce that residues other than cysteine are involved in the conversion of this enzyme into the oxidized form.