SITE-DIRECTED MUTAGENESIS OF FARNESYL DIPHOSPHATE SYNTHASE - EFFECT OF SUBSTITUTION ON THE 3 CARBOXYL-TERMINAL AMINO-ACIDS

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.