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-H-2]farnesyl diphosphate or (S)-[1-H-2]GGPP. The prenylated products were then degraded to dipeptides using carboxypeptidase Y. After HPLC purification, the prenylated dipeptide products were analyzed by H-1 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.