IDENTIFICATION OF SIGNIFICANT RESIDUES IN THE SUBSTRATE-BINDING SITE OF BACILLUS-STEAROTHERMOPHILUS FARNESYL DIPHOSPHATE SYNTHASE

Farnesyl diphosphate synthases have been shown to possess seven highly conserved regions (I-VII) in their amino acid sequences [Koyama et al . (1993) J. Biochem. (Tokyo) 113, 355-363]. Site-directed mutants of f arnesyl diphosphate synthase from Bacillus stearothermophilus were mad e to evaluate the roles of the conserved aspartic acids in region VI a nd lysines in regions I, V, and VI. The aspartate at position 224 was changed to alanine or glutamate (mutants designated as D224A and D224E , respectively); aspartates at positions 225 and 228 were changed to i soleucine and alanine (D225I, D228A); lysine at position 238 was chang ed to either alanine or arginine (K238A, K238R). The lysines at positi ons 47 and 183 were changed to isoleucine and alanine (K47I, K183A), r espectively. Kinetic analyses of the wild-type and mutant enzymes indi cated that the mutagenesis of Asp-224 and Asp-225 resulted in a decrea se of k(cat) values of approximately 10(4)- to 10(5)-fold compared to the wild type. On the other hand, D228A showed a k(cat) value approxim ately one-tenth of that of the wild type, and the K-m value for isopen tenyl diphosphate increased approximately 10-fold. Both K47I and K183A showed K-m values for isopentenyl diphosphate 20-fold larger and k(ca t) values 70-fold smaller than the wild type. These results suggest th at the two conserved lysines in regions I and V contribute to the bind ing of isopentenyl diphosphate and that the first and the second aspar tates in region VI are involved in catalytic function. Aspartate-228 i s also important for the binding of isopentenyl diphosphate rather tha n for catalytic reaction.