Yeast protein geranylgeranyltransferase type-I: Steady-state kinetics and substrate binding

Protein geranylgeranyltransferase type-I (PGGTase-I) catalyzes alkylation of the cysteine residue in proteins containing a consensus C-terminal CaaX sequence ending in Leu or Phe by the C-20 hydrocarbon moiety in geranylgeranyl diphosphate (GGPP). A kinetic study of the alkylation reaction was conducted with a continuous assay based on the fluorescence enhancement that accompanies geranylgeranylation of dansyl-GCIIL. The kinetic constants k(cat) = 0.34 +/- 0.01 s(-1), K-M(G) = 0.86 +/- 0.05 mu m for GGPP, and K-M(D) = 1.6 +/- 0.1 mu M for dansyl-GCIIL were calculated from initial rates measured at varying concentrations of the substrates. Inhibitor studies were conducted with dead-end inhibitors for GGPP and the peptide substrate. Double reciprocal plots for the peptide mimic Cys-AMBA-Leu gave a competitive pattern when plotted against varying concentrations of dansyl-GCIIL and an uncompetitive pattern against GGPP. Similar plots for 1-phosphono-(E,E,E)-geranylgeraniol, a dead-end inhibitor for GGPP, gave a competitive double reciprocal plot for varied concentrations of GGPP and induced potent substrate inhibition by dansyl-GCIIL when dansyl-GCIIL was the varied substrate. The dissociation constant (K-D) for the PGGTase-I.GGPP complex was 120 +/- 20 nM. These results are consistent with an ordered binding mechanism for PGGTase-I where GGPP adds before peptide.