Chen Annie P-C, Chen Yi-Hung, Liu Hsiao-Pei, Li Yu-Chin, Chen Chao-Tsen, Liang Po-Huang
Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.
J Am Chem Soc. 2002 Dec 25;124(51):15217-24. doi: 10.1021/ja020937v.
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.
法尼基焦磷酸（FPP）是许多参与类异戊二烯化合物生物合成的异戊烯基转移酶的常见底物。十一异戊烯焦磷酸合酶（UPPs）催化FPP链延长生成C(55)十一异戊烯焦磷酸（UPP），后者在细菌肽聚糖合成中作为脂质载体。在本研究中，制备了FPP的荧光类似物7-(2,6-二甲基-8-二磷酸-2,6-辛二烯氧基)-8-甲基-4-三氟甲基-色烯-2-酮香叶基焦磷酸，并用于研究其与大肠杆菌UPPs的配体相互作用。该化合物在336 nm处有最大吸收峰，在460 nm处有最大发射峰，不受蛋白质自发荧光的干扰。它是FPP的竞争性抑制剂（K(i) = 0.57 microM），也可作为替代底物（K(m) = 0.69 microM，k(cat) = 0.02 s(-)(1)），但主要与一个异戊烯基焦磷酸（IPP）反应，可能是由于产物转运不利。该化合物与酶结合时（化学计量比为1:1）荧光强度降低，通过FPP置换可恢复。使用停流装置测量了酶与该化合物的相互作用（k(on) = 55.3 microM(-)(1) s(-)(1)，k(off) = 31.6 s(-)(1)）。从竞争实验确定的产物解离速率常数（0.5 s(-)(1)）与我们之前动力学模拟的预测一致。与其他几种FPP解离缓慢的异戊烯基转移酶反应不同，UPPs以快速平衡的方式结合FPP，快速释放速率常数为30 s(-)(1)。本文介绍的FPP荧光类似物可能为研究一大类FPP结合蛋白的配体相互作用提供一种工具。
