Synthesis and Application of A Fluorescent Substrate Analogue to Study Ligand Interactions for Undecaprenyl Pyrophosphate Synthase Annie P.-C. Chen, ² Yi-Hung Chen, ‡,§ Hsiao-Pei Liu, ‡,§ Yu-Chin Li, § Chao-Tsen Chen,* ,§ and Po-Huang Liang* ,‡,² Contribution from the Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Institute of Biochemical Sciences, and Department of Chemistry, National Taiwan UniVersity, Taipei 10098, Taiwan Received July 9, 2002 Abstract: Farnesyl pyrophosphate (FPP) serves as a common substrate for many prenyltransferases involved in the biosynthesis of isoprenoid compounds. Undecaprenyl pyrophosphate synthase (UPPs) catalyzes the chain elongation of FPP to C55 undecaprenyl pyrophosphate (UPP) which acts as a lipid carrier in bacterial peptidoglycan synthesis. In this study, 7-(2,6-dimethyl-8-diphospho-2,6-octadienyloxy)- 8-methyl-4-trifluoromethyl-chromen-2-one geranyl pyrophosphate, a fluorescent analogue of FPP, was prepared and utilized to study ligand interactions with E. coli UPPs. This compound displays an absorbance maximum at 336 nm and emission maximum at 460 nm without interference from protein autofluorescence. It is a competitive inhibitor with respect to FPP (Ki ) 0.57 μM) and also serves as an alternative substrate (Km ) 0.69 μM and kcat ) 0.02 s -1 ), but mainly reacts with one isopentenyl pyrophosphate (IPP) probably due to unfavorable product translocation. Fluorescence intensity of this compound is reduced when bound to the enzyme (1:1 stoichiometry), and is recovered by FPP replacement. Using stopped-flow apparatus, the interaction of enzyme with the compound was measured (kon ) 55.3 μM -1 s -1 and koff ) 31.6 s -1 ). The product dissociation rate constant (0.5 s -1 ) determined from the competition experiments is consistent with our previous prediction from kinetic simulation. Unlike several other prenyltransferase reactions in which FPP dissociates slowly, UPPs binds FPP in a rapid equilibrium manner with a fast release rate constant of 30 s -1 . The fluorescent analogue of FPP presented here may provide a tool to investigate the ligand interactions for a broad class of FPP-binding proteins. Introduction Farnesyl pyrophosphate (FPP) serves as a branch point to synthesize a variety of natural isoprenoids. 1-3 This compound is synthesized by farnesyl pyrophosphate synthase (FPPs) via coupling of isopentenyl pyrophosphate (IPP) with its isomer, dimethylallyl pyrophosphate, followed by condensation of another IPP. 4,5 Starting from FPP, many linear isoprenoid compounds with various chain lengths are generated by multiple condensations with IPP catalyzed by a group of prenyltrans- ferases. 6,7 Undecaprenyl pyrophosphate synthase (UPPs) is one member of these prenyltransferases, which produces C 55 un- decaprenyl pyrophosphate (UPP) via consecutive condensation reactions of eight IPP with a FPP. The enzyme product acts as the carrier to transport lipid II to extracellular compartments for the synthesis of bacterial peptidoglycan. 8 Because of its pivotal role in cell wall biosynthesis, the enzyme is essential for bacterial survival and could be regarded as an antibiotic drug target. There is a need for convenient methodology to monitor its inhibitor binding activities. UPPs is the most well studied cis-prenyltransferase that shows no sequence homology with the trans enzymes such as FPPs (cis and trans enzymes catalyze the formation of cis and trans double bonds in the IPP condensation reactions, respectively). 9,10 We have previously determined rate constant for each IPP condensation catalyzed by UPPs using pre-steady-state kinetic approach. 11 However, the substrate and product dissociation kinetics were not examined due to the complexity of 8-step UPPs reaction and incapability of using isotope trapping * To whom correspondence should be addressed. Tel: 886-2-2785- 5696 ext. 6070. Fax: 886-2-2788-9759. E-mail: phliang@gate.sinica.edu.tw; Tel: 886-2-2364-5819. Fax: 886-2-2363-6359. E-mail: ctchen@ mail.ch.ntu.edu.tw. ² Institute of Biochemical Sciences, National Taiwan University. ‡ Institute of Biological Chemistry, Academia Sinica. § Department of Chemistry, National Taiwan University. (1) Kellogg, B. A.; Poulter, C. D. Curr. Opin. Chem. Biol. 1997, 1, 570-578. (2) Ogura, K.; Koyama, T.; Sagami, H. Subcellular Biochem. 1997, 28, chapter 3, 57-87. (3) Liang, P. H.; Ko, T. P.; Wang, A. H.-J. Eur. J. Biochem. 2002, 269, 3339- 3354. (4) Eberhardt, L.; Rilling, H. C. J. Biol. Chem. 1975, 250, 863-866. (5) Poulter, C. D.; Rilling, H. C. In Biosynthesis of Isoprenoid Compounds; Porter, J. W., Spurgeon, S. L., Eds.; Wiley: New York, 1981; Vol 1, pp 161-224. (6) Ogura, K.; Koyama, T. Chem. ReV. 1998, 98, 1263-1276. (7) Wang, K. C.; Ohnuma, S.-I. Biochim. Biophys. Acta 2000, 1529, 33-48. (8) Robyt, J. In Essentials of Carbohydrate Chemistry; Springer-Verlag: New York, 1998; Chapter 10, pp 305-318. (9) Shimizu, N.; Koyama, T.; Ogura, K. J. Biol. Chem. 1998, 273, 19 476- 19 481. (10) Apfel, C. M.; Takacs, B.; Fountoulakis, M.; Stieger, M.; Keck, W. J. Bacteriol. 1999, 181, 483-492. Published on Web 11/26/2002 10.1021/ja020937v CCC: $22.00 © 2002 American Chemical Society J. AM. CHEM. SOC. 2002, 124, 15217-15224 9 15217