Chemical modification of human UDP-glucuronosyltransferase UGT1*6 by diethyl pyrocarbonate: possible involvement of a histidine residue in the catalytic process.
Chemical modification of human UDP-glucuronosyltransferase UGT1*6 by diethyl pyrocarbonate: possible involvement of a histidine residue in the catalytic process.
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焦碳酸二乙酯对人 UDP-葡萄糖醛酸基转移酶 UGT1*6 的化学修饰：组氨酸残基可能参与催化过程。
DOI：
10.1006/abbi.1994.1112
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发表时间：
1994
影响因子：
3.9
通讯作者：
J. Magdalou
中科院分区：
文献类型：
--
作者：
E. Battaglia;M. Pritchard;M. Ouzzine;S. Fournel‐Gigleux;A. Radominska;G. Siest;J. Magdalou
用焦碳酸二乙酯（DEPC）对V79细胞系富集膜组分中的重组人肝UDP-葡萄糖醛酸基转移酶UGT 1 *6进行化学修饰，导致葡萄糖醛酸化反应快速失活，用4-甲基伞形酮作为糖苷配基底物测定，在pH 6.0和25 ℃下的二级速率常数为3110 M-1.min-1。羟胺可使酶活性恢复。用0.2 mM DEPC化学修饰60 s使表观Vmax降低2.4倍，而不显著影响对4-甲基伞形酮和UDP-葡萄糖醛酸的表观Km。类似地，光活化辅底物类似物[β-32 P]5-叠氮基-UDP-葡萄糖醛酸与活性位点的结合不受化学修饰的影响。该酶的保护，防止这种失活的4-甲基伞形酮，这表明修改后的残基位于或附近的糖苷配基结合位点。相反，共底物UDP-葡萄糖醛酸增强了不可逆的抑制，表明结合后蛋白质的构象变化。失活的pH依赖性与pKa为6.1的氨基酸残基的修饰一致。另一方面，分析葡萄糖醛酸化反应的Vmax和Vmax/Km值随pH值的变化，发现存在两个pKa在5.7-6.0范围内的必需残基。重组酶的化学修饰数据以及活性的pH依赖性数据强烈表明涉及组氨酸残基，其对DEPC具有高度反应性，可能是人UGT 1 *6支持的葡萄糖醛酸化反应的碱催化剂。
Chemical modification with diethyl pyrocarbonate (DEPC) of the recombinant human liver UDP-glucuronosyltransferase UGT1*6 in enriched membrane fractions from a V79 cell line resulted in a rapid inactivation of the glucuronidation reaction, measured with 4-methyl-umbelliferone as aglycone substrate, with a second-order rate constant of 3110 M-1.min-1 at pH 6.0 and 25 degrees C. The enzymatic activity was restored by hydroxylamine. Chemical modification with 0.2 mM DEPC for 60 s decreased the apparent Vmax 2.4-fold without significantly affecting the apparent Km toward 4-methylumbelliferone and UDP-glucuronic acid. Similarly, the binding of the photoactivatable cosubstrate analog [beta-32P]5-azido-UDP-glucuronic acid to the active site was not affected by the chemical modification. The enzyme was protected against this inactivation by 4-methylumbelliferone, suggesting that the modified residue was located in or near the aglycone binding site. In contrast, the cosubstrate UDP-glucuronic acid potentiated the irreversible inhibition, indicating a conformational change in the protein upon binding. The pH-dependence of the inactivation was in agreement with the modification of an amino acid residue with a pKa of 6.1. On the other hand, analysis of the variation of Vmax and Vmax/Km values of the glucuronidation reaction as a function of the pH revealed the presence of two essential residues with a pKa within the range 5.7-6.0. The data of the chemical modification of the recombinant enzyme together with that of the pH dependence of the activity strongly suggest the involvement of a histidine residue, highly reactive toward DEPC, which could be the base catalyst of the glucuronidation reaction supported by human UGT1*6.