YEAST PROTEIN FARNESYLTRANSFERASE - STEADY-STATE KINETIC-STUDIES OF SUBSTRATE-BINDING

Protein farnesyltransferase (PFTase) catalyzes the alkylation of cyste ine in C-terminal CaaX sequences of a variety of proteins, including R as, nuclear lamins, large G-proteins, and phosphodiesterases, by farne syl diphosphate (FPP). These modifications enhance the ability of the proteins to associate with membranes and are essential for their respe ctive functions. The binding mechanism for yeast PFTase was deduced fr om a combination of steady-state kinetic and equilibrium studies. Rate s for prenylation were measured by a continuous assay based on an enha ncement in the fluorescence of the dansyl moiety in pentapeptide dansy l-GCVIA upon farnesylation by FPP. Unreactive substrate analogs for FP P and dansyl-GCVIA gave steady-state inhibition patterns for the dead- end inhibitors typical of an ordered sequential mechanism in which FPP adds to the enzyme before the peptide. The kinetic analysis was compl icated by substrate inhibition for dansyl-GCVIA. The substrate inhibit ion was reversed at high concentrations of FPP, indicating that format ion of the nonproductive enzyme-peptide complex is competitive with re spect to FPP. Progress curves were fitted to an integrated form of the rate expression to determine the catalytic constant, k(cat) = 4.5 +/- 1.9 s(-1), and the Michaelis constant for dansyl-GCVIA, K-M(D) = 0.9 +/- 0.1 mu M. The dissociation constant for FPP, K-D = 75 +/- 15 nM, w as measured using a membrane retention assay.