Insight into the activation mechanism of Escherichia coli octaprenyl pyrophosphate synthase derived from pre-steady-state kinetic analysis Jian-Jung Pan a , Tun-Hsun Kuo b , Yi-Kai Chen a , Lee-Wei Yang a , Po-Huang Liang a;b; * a Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan b Institute of Biochemical Sciences, National Taiwan University, Taipei 10098, Taiwan Received 26 July 2001; received in revised form 11 September 2001; accepted 13 September 2001 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 31 ) was 100 times larger than the steady-state rate (0.02 s 31 ). The high molecular weight fractions and Triton could accelerate the steady-state rate by 3-fold (0.06 s 31 ) but insufficient to cause full activation (100-fold). A burst product formation was observed in enzyme multiple turnovers indicating a slow product release. ß 2002 Elsevier Science B.V. All rights reserved. Keywords : Pre-steady-state kinetics ; Prenyltransferase ; Rapid quench ; Single turnover 1. Introduction Isoprenoid compounds are widely distributed in nature and serve a variety of important biological functions [1]. Using a ¢ve-carbon isopentenyl pyro- phosphate (IPP) as the building block, isoprenoids are synthesized by a group of enzymes called prenyl- transferases, that catalyze the multiple IPP conden- sation reactions with allylic pyrophosphate for chain elongation. The prenyltransferase reaction results in a trans or cis double bond during each IPP conden- sation and the enzymes were thus classi¢ed as Z- and E-type, respectively. In general, Z-type prenyltrans- ferases catalyze the formation of long-chain poly- 0167-4838 / 02 / $ ^ see front matter ß 2002 Elsevier Science B.V. All rights reserved. PII:S0167-4838(01)00283-7 Abbreviations : IPP, isopentenyl pyrophosphate ; FPP, farnesyl pyrophosphate ; UPPs, undecaprenyl pyrophosphate synthase ; UPP, undecaprenyl pyrophosphate ; OPPs, octaprenyl pyrophos- phate synthase ; OPP, octaprenyl pyrophosphate ; SPPs, solanesyl pyrophosphate synthase ; HMF, high molecular weight fraction ; BSA, bovine serum albumin; PCR, polymerase chain reaction; NiNTA, nickel nitrilotriacetic acid; Tris, tris(hydroxymethyl)ami- nomethane ; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesul- fonic acid; EDTA, ethylenediaminetetraacetic acid; SDS^ PAGE, sodium dodecyl sulfate^polyacrylamide gel electrophore- sis ; TLC, thin layer chromatography * Corresponding author, at address a. Fax : +886-2-2788-97-59. E-mail address : phliang@gate.sinica.edu.tw (Po-Huang Liang). Biochimica et Biophysica Acta 1594 (2002) 64^73 www.bba-direct.com