T. Koyama et al., STRUCTURAL AND FUNCTIONAL ROLES OF THE CYSTEINE RESIDUES OF BACILLUS-STEAROTHERMOPHILUS FARNESYL DIPHOSPHATE SYNTHASE, Biochemistry, 33(42), 1994, pp. 12644-12648
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.