Stereochemical analysis of the reaction catalyzed by human protein geranylgeranyl transferase.
Protein geranylgeranyltransferase type I (PGGTase-I) catalyzes the nucleophilic substitution reaction between the C(20) geranylgeranyl diphosphate (GGPP) and a protein-derived thiol to form a thioether linkage. Here, we describe the stereochemical outcome, at the isoprenoid C1, of the reaction catalyzed by human PGGTase-I. To accomplish this, the pentapeptide N-dansyl-GCVLL was first enzymatically prenylated by human PGGTase-I with either (S)-[1-(2)H]farnesyl diphosphate or (S)-[1-(2)H]GGPP. The prenylated products were then degraded to dipeptides using carboxypeptidase Y. After HPLC purification, the prenylated dipeptide products were analyzed by (1)H NMR spectroscopy. The final spectra were compared with the spectra from the same product obtained via chemical synthesis to deduce the stereochemistry of the PGGTase-I-catalyzed reaction. This comparison showed that the reaction proceeds with inversion of configuration with no detectable (< 6%) racemization. These results are more consistent with an associative-type mechanism, but they cannot be used to rule out a dissociative mechanism involving a rigid, solvent-sequestered, tight ion pair.
Clausen
V A
VA
Department of Chemistry, University of Minnesota, Minneapolis 55455, USA.
Edelstein
R L
RL
Distefano
M D
MD
D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D003619:Dansyl Compounds / Q000138:chemical synthesis; D003903:Deuterium / Q000737:chemistry; D004151:Dipeptides / Q000138:chemical synthesis; D004224:Diterpenes / Q000737:chemistry; D005204:Farnesol / Q000031:analogs & derivatives / Q000737:chemistry; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D009842:Oligopeptides / Q000138:chemical synthesis; D017368:Protein Prenylation; D013237:Stereoisomerism; D013379:Substrate Specificity
D003619:Dansyl Compounds; D004151:Dipeptides; D004224:Diterpenes; D009842:Oligopeptides; D005204:Farnesol; C017338:geranylgeraniol; D003903:Deuterium; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I