Structural and functional roles of the cysteine residues of Bacillus stearothermophilus farnesyl diphosphate synthase.
p-(Chloromercuri)benzoic acid inhibited the catalytic activity of Bacillus 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 Escherichia coli and purified to homogeneity. All of them were active as farnesyl diphosphate synthase, showing specific activities comparable to that of the wild-type enzyme. These results indicate that neither of the cysteines is essential for catalytic function. The C73F mutant, however, was very sensitive to heat treatment, while C73S was as highly stable as the wild type. The Km value of C289F for isopentenyl diphosphate is 10 times that of the wild type. The wild-type enzyme was converted into an oxidized form which was separable from the native enzyme on ion-exchange chromatography, and this conversion was accelerated by cupric ions. Similar conversion has previously been reported by several researchers, who found the occurrence of two forms of pig liver FPP synthase and who attributed this phenomenon to the oxidoreduction of sulfhydryl and disulfide groups. However, even the C73S-C289S mutant, which has no cysteine residues, was also converted into an oxidized form as in the case of the wild-type enzyme. These results provide evidence that residues other than cysteine are involved in the conversion of this enzyme into the oxidized form.
Koyama
T
T
Institute for Chemical Reaction Science, Tohoku University, Miyagi, Japan.
Obata
S
S
Saito
K
K
Takeshita-Koike
A
A
Ogura
K
K
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D001483:Base Sequence; D002729:Chloromercuribenzoates; D003545:Cysteine / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D051228:Geranyltranstransferase; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000378:metabolism; D014166:Transferases / Q000737:chemistry* / Q000378:metabolism; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D011994:Recombinant Proteins; D020245:p-Chloromercuribenzoic Acid; D014166:Transferases; D019883:Alkyl and Aryl Transferases; D051228:Geranyltranstransferase; D003545:Cysteine