2-cyclopropylideneethyl analogues of prenyl pyrophosphates and their interactions with some prenyltransferases

We have investigated the enzymatic behavior of several analogues of prenyl pyrophosphates that were designed to assess the lifetime of the initial carbocationic intermediates that are alleged to form during enzymatic prenyl group transfer. Each of these analogues was designed to undergo a rapid reaction, upon formation of the initial allylic carbocation, that would divert an enzymatic reaction from its normal course and lead to new products. Compounds of the first set, novel analogues of farnesyl pyrophosphate bearing a cyclopropylideneethyl group in place of the proximal isoprene unit, are competitive inhibitors against PFTase with IC50 values of 0.7 muM and 3.5 muM, respectively. The complete absence of time-dependent irreversible inhibition by these analogues suggests that the reaction may have considerably more associative character than is indicated by earlier studies. This led us to investigate farnesyl pyrophosphate synthase, which catalyzes prenyl group transfer to a more weakly nucleophilic substrate, with hopes that the mechanism would exhibit more dissociative character. These analogues of dimethylallyl pyrophosphate did not lead to irreversible inhibition, and analysis of the reaction mixture by 1H-NMR spectroscopy also indicated the absence of diverted product. Since the non-enzymatic rate of the diversionary electrocyclic reaction can be measured by rapid chemical kinetics, our measurements impose strict limits on the kinetic lifetime of the purported, initially formed enzymatic allylic cation.