Product distribution and pre-steady-state kinetic analysis of Escherichia coli undecaprenyl pyrophosphate synthase reaction

Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the condensation of ei ght molecules of isopentenyl pyrophosphate (IPP) with farnesyl pyrophosphat e (FPP) to generate C-55 undecaprenyl pyrophosphate. We investigated the ki netics and mechanism of this reaction pathway using Escherichia coli UPPs. With a variety of different ratios of enzyme to substrate and FPP to IPP in the presence or absence of Triton, different product distributions were fo und. In the presence of excess FPP, the intermediates (C-25-C-50) accumulat ed. Under a condition with enzyme and FPP in excess of IPP, instead of C-20 -geranylgeranyl pyrophosphate, C-20, C-25, and C-30 were the major products . The UPPs steady-state k(cat) value (2.5 s(-1)) in the presence of 0.1% Tr iton was 190-fold larger than in the absence of Triton (0.013 s(-1)). The k (cat) value matched the rate constant of each IPP condensation obtained fro m the enzyme single-turnover experiments. This suggested that the IPP conde nsation rather than product release was the rate-limiting step in the prese nce of Triton. In the absence of Triton, the intermediates formed and disap peared in a similar manner under enzyme single turnover in contrast to the slow steady-state rate, which indicated a step after product generation was rate limiting, This was further supported by a burst product formation. Ju dging from the accumulation level of C-55, C-60, and C-65, their dissociati on from the enzyme cannot be too slow and an even slower enzyme conformatio nal change with a rate of 0.001 s(-1) might govern the UPPs reaction rate u nder the steady-state condition in the absence of Triton.