Properties and kinetic mechanism of recombinant mammalian protein geranylgeranyltransferase type I.
Protein geranylgeranyltransferase type I (GGTase I) catalyzes the prenylation of a number of proteins that play important roles in cellular signaling. The recent cDNA cloning of this enzyme (Zhang, F. L., Diehl, R. E., Kohl, N. E., Gibbs, J. B., Giros, B., Casey, P. J., and Omer, C. A. (1994) J. Biol. Chem. 269, 3175-3180) has allowed us to develop an expression system for obtaining large quantities of the enzyme. Co-infection of insect cells with recombinant baculoviruses encoding the two subunits of the enzyme results in GGTase I accumulation within the cells to levels of > 20% of cytosolic protein. The recombinant enzyme could be readily purified by ion-exchange chromatography and is shown to possess the activity and specificity of the enzyme obtained from mammalian tissues. Production of the recombinant enzyme allowed us to confirm its identity as a zinc metalloenzyme by direct identification of the metal using atomic absorption spectroscopy. We also identify two substrate analogs that are competitive inhibitors of GGTase I. One is a novel isoprenoid analog, 3-aza-GGPP, which inhibits the enzyme with a Ki of 15 nM. The second inhibitor is the tetrapeptide Cys-Val-Phe-Leu, which exhibits a Ki of 50 nM. The use of these inhibitors, coupled with a steady-state kinetic analysis of the enzyme, reveals that the reaction catalyzed by GGTase I proceeds through a random ordered sequential mechanism.
Zhang
F L
FL
Department of Molecular Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710.
Moomaw
J F
JF
Casey
P J
PJ
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D000818:Animals; D007700:Kinetics; D008969:Molecular Sequence Data; D051381:Rats; D011994:Recombinant Proteins; D013379:Substrate Specificity; D014166:Transferases / Q000378:metabolism*; D015032:Zinc / Q000737:chemistry
D011994:Recombinant Proteins; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; D015032:Zinc