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 C20 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 microM for GGPP, and K(M)(D) = 1.6 +/- 0.1 microM 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 x GGPP complex was 120 +/- 20 nM. These results are consistent with an ordered binding mechanism for PGGTase-I where GGPP adds before peptide.
Stirtan
W G
WG
Department of Chemistry, University of Utah, Salt Lake City 84112, USA.
Poulter
C D
CD
D019883:Alkyl and Aryl Transferases; D001667:Binding, Competitive; D003619:Dansyl Compounds / Q000494:pharmacology; D004224:Diterpenes / Q000494:pharmacology; D007700:Kinetics; D009842:Oligopeptides / Q000494:pharmacology; D017368:Protein Prenylation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013379:Substrate Specificity; D014166:Transferases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D003619:Dansyl Compounds; D004224:Diterpenes; D009842:Oligopeptides; C017338:geranylgeraniol; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I