Involvement of Ser-451 and Ser-452 in the Catalysis of Human γ-Glutamyl Transpeptidase (*)-猫眼课题宝

Ser-451 和 Ser-452 参与人 γ-谷氨酰转肽酶的催化 (*)

DOI：

发表时间：

1995

期刊：

Journal of Biological Chemistry

影响因子：

4.8

通讯作者：

中科院分区：

生物学2区

文献类型：

作者：

Y. Ikeda;J. Fujii;Mary E. Anderson;Naoyuki Taniguchi;A. Meister

通过对人、大鼠、猪和两种细菌γ-谷氨酰转肽酶轻亚基的序列比对，对保守的丝氨酸残基进行定点突变，确定了催化γ-谷氨酰转肽酶所需的丝氨酸残基。使用杆状病毒-昆虫细胞系统表达这些丝氨酸残基被Ala取代的重组人γ-谷氨酰转肽酶。在Ser-385、-413或-425位取代Ala，几乎可以产生完全活性的酶。然而，在Ser-451或-452处替换Ala产生的酶的活性仅为野生型酶的1%左右。此外，其双突变体的活性仅为野生型的0.002%。使用甘氨酰甘氨酸作为受体的转肽动力学分析表明，Ser-451和−452突变体的Vmax值显著降低（约为野生型的3%）;然而，它们对L-γ-谷氨酰-对硝基苯胺作为供体的Km值仅比野生型增加约5倍。Ser-451和-452的双重突变进一步降低了Vmax值，仅为野生型的0.005%，而这种突变对供体的Km值仅产生轻微影响（增加2倍）。L-γ-谷氨酰-对硝基苯胺在突变体中的水解反应动力学值遵循与转肽反应相似的趋势。Ser-451、-452和它们的双突变体酶被一种有效的抑制剂acivicin灭活的速率小于野生型酶的1%。作为γ-谷氨酰基团的竞争性抑制剂的L-丝氨酸的双突变体的Ki值仅比野生型增加9倍，而作为抑制性过渡态类似物的丝氨酸-硼酸盐复合物的Ki比野生型酶高1,000倍以上。这些结果表明，Ser-451和−452都位于能够与γ-谷氨酰基相互作用并参与催化的位置，可能作为亲核试剂或通过稳定过渡态。

The serine residue required for catalysis of γ-glutamyl transpeptidase was identified by site-specific mutagenesis of the conserved serine residues on the basis of sequence alignment of the light subunit of human, rat, pig and two bacterial enzymes. Recombinant human γ-glutamyl transpeptidases with replacements of these serine residues by Ala were expressed using a baculovirus-insect cell system. Substitutions of Ala at Ser-385, −413 or −425 yielded almost fully active enzymes. However, substitutions of Ala at Ser-451 or −452 yielded enzymes that were only about 1% as active as the wild-type enzyme. Further, their double mutant is only 0.002% as active as the wild type. Kinetic analysis of transpeptidation using glycylglycine as acceptor indicates that the Vmax values of Ser-451 and −452 mutants are substantially decreased (to about 3% of the wild type); however, their Km values for L-γ-glutamyl-p-nitroanilide as donor were only increased about 5 fold compared to that of the wild type. The double mutation of Ser-451 and −452 further decreased the Vmax value to only about 0.005% of the wild type, while this mutation produced only a minor effect (2-fold increase) on the Km value for the donor. The kinetic values for the hydrolysis reaction of L-γ-glutamyl-p-nitroanilide in the mutants followed similar trends to those for transpeptidation. The rates of inactivation of Ser-451, −452 and their double mutant enzymes by acivicin, a potent inhibitor, were less than 1% that of the wild-type enzyme. The Ki value of the double mutant for L-serine as a competitive inhibitor of the γ-glutamyl group is only 9 fold increased over that of the wild type, whereas the Ki for the serine-borate complex, which acts as an inhibitory transition-state analog, was more than 1,000 times higher than for the wild-type enzyme. These results suggest that both Ser-451 and −452 are located at the position able to interact with the γ-glutamyl group and participate in catalysis, probably as nucleophiles or through stabilization of the transition state.