posted on 2001-03-09, 00:00authored byValerie A. Clausen, Rebecca L. Edelstein, Mark D. Distefano
Protein geranylgeranyltransferase type I (PGGTase-I) catalyzes the nucleophilic substitution
reaction between the C20 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)-[1-2H]farnesyl diphosphate or (S)-[1-2H]GGPP. The
prenylated products were then degraded to dipeptides using carboxypeptidase Y. After HPLC purification,
the prenylated dipeptide products were analyzed by 1H 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.