T. Koyama et al., SITE-DIRECTED MUTAGENESIS OF FARNESYL DIPHOSPHATE SYNTHASE - EFFECT OF SUBSTITUTION ON THE 3 CARBOXYL-TERMINAL AMINO-ACIDS, Canadian journal of chemistry, 72(1), 1994, pp. 75-79
Site-directed mutation was introduced into the gene for the farnesyl d
iphosphate synthase of Bacillus stearothermophilus. To investigate the
significance of the three C-terminal amino acids, where arginine is c
ompletely conserved throughout the farnesyl diphosphate synthases of p
rokaryotes and eukaryotes, three kinds of mutant enzymes, R295V, D296G
, and H297L, which have replacements of arginine-295 with valine, aspa
rtate-296 with glycine, and histidine-297 with leucine, respectively,
were over-produced and purified to homogeneity. All of the three mutan
t enzymes showed similar catalytic activities to that of the wild-type
enzyme, indicating that the basic amino acids including the conserved
arginine in the C-terminal region are not essential for catalytic fun
ction. They were also similar to the wild-type enzyme with respect to
pH optima, thermostability, reaction product, and kinetic parameters f
or allylic substrates. However, their K-m values for isopentenyl dipho
sphate are approximately twice that of the wild type.