Vashishtha S C, Hawes E M, McKay G, McCann D J
Drug Metabolism and Drug Disposition Group, College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Canada.
Drug Metab Dispos. 2001 Oct;29(10):1290-5.
A series of eight 1-substituted imidazoles was investigated as model substrates for glucuronidation at an aromatic tertiary amine of polyaza heterocyclic ring systems. The human UDP-glucuronosyltransferases (UGTs) involved and substrate specificities were investigated. Nine expressed enzymes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A9, UGT1A10, UGT2B7, and UGT2B15) were examined, but only UGT1A4 catalyzed the formation of a quaternary ammonium-linked glucuronide metabolite for six of the substrates. UGT1A3 also catalyzed the glucuronidation of the previously investigated 1-phenylimidazole but none of the newly investigated compounds. No glucuronidation was observed with 1-(4-nitrophenyl)imidazole, the compound with the 4-phenyl substituent with the largest electron withdrawing effect. The incubation conditions for the determination of the kinetic constants for UGT1A4 catalysis of six substrates were optimized and included incubation at pH 7.4 with alamethicin at 10 microg/mg of protein. Latency disrupting agents, including alamethicin and sonication, enhanced glucuronidation 1.25-fold at most. There were 17.5- and 2.2-fold variations in the apparent K(m) (range, 0.18-3.15 mM) and V(max) values (range, 0.16-0.35 nmol/min/mg of protein). Linear correlation analyses between UGT1A4 kinetics and substrate physicochemical parameters showed significant correlation between V(max) and both the partition coefficient (log P, n-octanol/water) and pK(a) and between K(m) and pK(a), thereby indicating that the lipophilicity and the ease of availability of the tertiary amine lone pair of electrons of the substrate are important with respect to enzyme catalysis.
研究了一系列8种1-取代咪唑作为聚氮杂杂环系统芳香叔胺葡萄糖醛酸化的模型底物。研究了所涉及的人尿苷二磷酸葡萄糖醛酸基转移酶（UGTs）和底物特异性。检测了9种表达的酶（UGT1A1、UGT1A3、UGT1A4、UGT1A6、UGT1A7、UGT1A9、UGT1A10、UGT2B7和UGT2B15），但只有UGT1A4催化了6种底物形成季铵连接的葡萄糖醛酸代谢物。UGT1A3也催化了先前研究的1-苯基咪唑的葡萄糖醛酸化，但对新研究的化合物均无催化作用。对于具有最大吸电子效应的4-苯基取代基的化合物1-（4-硝基苯基）咪唑，未观察到葡萄糖醛酸化。优化了用于测定UGT1A4催化6种底物动力学常数的孵育条件，包括在pH 7.4下用阿拉霉素以10μg/mg蛋白质进行孵育。包括阿拉霉素和超声处理在内的潜伏期破坏剂最多可使葡萄糖醛酸化增强1.25倍。表观K(m)（范围为0.18 - 3.15 mM）和V(max)值（范围为0.16 - 0.35 nmol/min/mg蛋白质）分别有17.5倍和2.2倍的变化。UGT1A4动力学与底物物理化学参数之间的线性相关分析表明，V(max)与分配系数（log P，正辛醇/水）和pK(a)之间以及K(m)与pK(a)之间存在显著相关性，从而表明底物的亲脂性和叔胺孤对电子的可利用性对于酶催化很重要。
