PROTEIN FARNESYLTRANSFERASE - KINETICS OF FARNESYL PYROPHOSPHATE BINDING AND PRODUCT RELEASE

Protein farnesyltransferase (FTase) catalyzes the prenylation of Ras a nd several other key proteins involved in cell regulation. The mechani sm of the FTase reaction was elucidated by pre-steady-state and steady -state kinetic analysis. FTase catalyzed the farnesylation of biotinyl ated peptide substrate (BiopepSH) by farnesyl pyrophosphate (FPP) to a n S-farnesylated peptide (BiopepS-C-15). The steady state kinetic mech anism was ordered. FTase bound FPP in a two-step process with an effec tive dissociation rate constant of 0.013 s(-1) and an overall K-d of 2 .8 nM. BiopepSH reacted with FTase . FPP irreversibly, with a second-o rder rate constant of 2.2 x 10(5) M(-1) s(-1), to form FTase . BiopepS -C-15. Because most of the FPP in FTase FPP was trapped as FTase . Bio pepS-C-15 at high concentrations of BiopepSH, FPP dissociated slowly f rom the ternary complex relative to catalysis, so that the commitment to catalysis was high, The maximal rate constant for formation of FTas e . BiopepS-C-15 (enzyme-bound product) is much larger than k(cat) (0. 06 s(-1)), indicating that product release is the rate-determining ste p in the reaction mechanism.