Senay C, Ouzzine M, Battaglia E, Pless D, Cano V, Burchell B, Radominska A, Magdalou J, Fournel-Gigleux S
Unité de Recherche Associé au Centre National de la Recherche Scientifique 597, Université Henri Poincaré Nancy 1, France.
Mol Pharmacol. 1997 Mar;51(3):406-13.
The hepatic UDP-glucuronosyltransferase UGT16 is actively involved in the glucuronidation of short and planar phenols in humans. Based on the irreversible inhibition of the enzyme on chemical modification by 2,3-butanedione and diethyl pyrocarbonate, the roles of His54 and Arg52 were investigated by oligonucleotide site-directed mutagenesis. These amino acids belong to a consensus sequence LX2-R52-G-H54-X3-V-L located in a conserved hydrophobic region of the variable amino-terminal domain of UGT. Arg52 was replaced by alanine (mutant R52A), and His54 was replaced by alanine or glutamine (mutants H54A and H54Q). The immunological and catalytic properties of UGT16 and mutants were examined after stable expression in V79 cell lines. Immunoblots and immunoprecipitation studies revealed that the mutant and UGT16 proteins were expressed in the microsomal membranes in similar amounts. However, replacement of His54 by glutamine led to a complete loss of activity toward 4-methylumbelliferone, and the Vmax value was decreased 4-5-fold in the mutants R52A and H54A compared with the wild-type enzyme. The dissociation constants that characterize the binding of 4-methylumbelliferone and UDP-glucuronic acid to UGT16 were not greatly affected by the mutations. Interestingly, H54Q was not recognized by specific antibodies to the amino-terminal portion of UGT16, thereby indicating that this amino acid was critical to antibody recognition. In contrast, the mutants R52A and H54A could not be differentiated from the wild-type protein by pH optimum or thermal denaturation. Furthermore, these mutants were still sensitive to irreversible inhibition by diethyl pyrocarbonate and 2,3-butanedione, with second-order inactivation constant values similar to those obtained for UGT16. Altogether, the strict conservation of His54 and Arg52 and the mutational analysis of these residues suggest that these amino acids in the hydrophobic amino-terminal consensus sequence LX2-R52-G-H54-X3-V-L are important for the function and the structure required for optimal catalytic efficiency of UGT1*6.
肝脏UDP - 葡萄糖醛酸基转移酶UGT16积极参与人体中短链和平面酚类的葡萄糖醛酸化反应。基于2,3 - 丁二酮和焦碳酸二乙酯对该酶化学修饰的不可逆抑制作用，通过寡核苷酸定点诱变研究了His54和Arg52的作用。这些氨基酸属于位于UGT可变氨基末端结构域保守疏水区域的共有序列LX2 - R52 - G - H54 - X3 - V - L。将Arg52替换为丙氨酸（突变体R52A），将His54替换为丙氨酸或谷氨酰胺（突变体H54A和H54Q）。在V79细胞系中稳定表达后，检测了UGT16及其突变体的免疫学和催化特性。免疫印迹和免疫沉淀研究表明，突变体和UGT16蛋白在微粒体膜中的表达量相似。然而，用谷氨酰胺替换His54导致对4 - 甲基伞形酮的活性完全丧失，与野生型酶相比，突变体R52A和H54A的Vmax值降低了4 - 5倍。表征4 - 甲基伞形酮和UDP - 葡萄糖醛酸与UGT16结合的解离常数受突变影响不大。有趣的是，H54Q不被针对UGT16氨基末端部分的特异性抗体识别，从而表明该氨基酸对抗体识别至关重要。相比之下，突变体R52A和H54A在最适pH或热变性方面与野生型蛋白无法区分。此外，这些突变体对焦碳酸二乙酯和2,3 - 丁二酮的不可逆抑制仍敏感，二级失活常数与UGT16的相似。总之，His54和Arg52的严格保守以及这些残基的突变分析表明，疏水氨基末端共有序列LX2 - R52 - G - H54 - X3 - V - L中的这些氨基酸对于UGT1*6的功能以及最佳催化效率所需的结构很重要。
