Synthesis and Application of A Fluorescent Substrate Analogue to Study Ligand Interactions for Undecaprenyl Pyrophosphate Synthase
Resource
Journal of the American Chemical Society 124 (51): 15217-15224
Journal
Journal of the American Chemical Society
Journal Volume
124
Journal Issue
51
Pages
15217-15224
Date Issued
2002
Date
2002
Author(s)
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 C(55) 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 (K(i) = 0.57 microM) and also serves as an alternative substrate (K(m) = 0.69 microM and k(cat) = 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 (k(on) = 55.3 microM(-)(1) s(-)(1) and k(off) = 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.
Type
journal article
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