Insight into the activation mechanism of Escherichia coli octaprenyl pyrophosphate synthase derived from pre-steady-state kinetic analysis.
Octaprenyl pyrophosphate synthase (OPPs) catalyzes the sequential condensation of five molecules of isopentenyl pyrophosphate with farnesyl pyrophosphate to generate all-trans C40-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.
Pan
Jian-Jung
JJ
Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
Kuo
Tun-Hsun
TH
Chen
Yi-Kai
YK
Yang
Lee-Wei
LW
Liang
Po-Huang
PH
D019883:Alkyl and Aryl Transferases / Q000378:metabolism*; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D045782:Hemiterpenes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009943:Organophosphorus Compounds / Q000378:metabolism; D011092:Polyethylene Glycols; D011106:Polyisoprenyl Phosphates / Q000096:biosynthesis* / Q000378:metabolism; D012717:Sesquiterpenes; D013696:Temperature
D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C004809:isopentenyl pyrophosphate; C008981:polyprenylpyrophosphate; D011092:Polyethylene Glycols; C004808:farnesyl pyrophosphate; D019883:Alkyl and Aryl Transferases; C048987:octaprenyl pyrophosphate synthetase