Insight into the activation mechanism of Escherichia coli octaprenyl pyrophosphate synthase derived from pre-steady-state kinetic analysis

Abstract

Octaprenyl pyrophosphate synthase (OPPs) catalyzes the sequential condensation of five molecules of isopentenyl pyrophosphate with farnesyl pyrophosphate to generate all- trans C 40-octaprenyl pyrophosphate, which constitutes the side chain of ubiquinone. Due to the slow product release, a long-chain polyprenyl pyrophosphate synthase often requires detergent or another factor for optimal activity. Our previous studies in examining the activity enhancement of Escherichia coli undecaprenyl pyrophosphate synthase have demonstrated a switch of the rate-determining step from product release to isopentenyl pyrophosphate (IPP) condensation reaction in the presence of Triton [12]. In order to understand the mechanism of enzyme activation for E. coli OPPs, a single-turnover reaction was performed and the measured IPP condensation rate (2 s −1) was 100 times larger than the steady-state rate (0.02 s −1). The high molecular weight fractions and Triton could accelerate the steady-state rate by 3-fold (0.06 s −1) but insufficient to cause full activation (100-fold). A burst product formation was observed in enzyme multiple turnovers indicating a slow product release.