Mutational analysis of the thyrotropin-releasing hormone-degrading ectoenzyme. similarities and differences with other members of the M1 family of aminopeptidases and thermolysin.
Mutational analysis of the thyrotropin-releasing hormone-degrading ectoenzyme. similarities and differences with other members of the M1 family of aminopeptidases and thermolysin.
复制标题
促甲状腺素释放激素降解胞外酶的突变分析。
DOI：
10.1021/bi010695w
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
K. Bauer
中科院分区：
文献类型：
--
作者：
Theofilos G. Papadopoulos;J. A. Kelly;K. Bauer
促甲状腺激素释放肽降解胞外酶（TRH-DE）是一种TRH特异性肽酶，催化肽能信号物质TRH的失活。如序列比对所示，TRH-DE和氨肽酶M1家族的其他成员具有一组独特的共同保守氨基酸残基。通过替换参与催化作用的puristic氨基酸残基，我们可以证明突变体E408 D、E442 D、E464 Q、E464 D、Y 528 F、H507 R和H507 F的酶活性显著降低，主要是由于V（max）的变化。突变体酶E408 Q和E442 Q是无活性的，而突变体R488 Q、R488 A和Y554 F的比酶活性与野生型酶相似。这些数据有力地表明，E408，E442，Y 528和H507参与TRH-DE的催化过程，而E464可能代表第三个锌配位残基，可能相当于嗜热菌蛋白酶中的E166。相反，氨基酸残基R488和Y554似乎不参与TRH-DE的催化机制。
Thyrotropin-releasing hormone-degrading ectoenzyme (TRH-DE) is a TRH-specific peptidase which catalyzes the inactivation of the peptidergic signal substance TRH. As indicated by sequence alignment, TRH-DE and the other members of the M1 family of aminopeptidases have a distinct set of conserved amino acid residues in common. By replacing amino acid residues that are putatively involved in catalysis, we could demonstrate that the enzymatic activities of the mutants E408D, E442D, E464Q, E464D, Y528F, H507R, and H507F are dramatically decreased, essentially due to the changes of V(max). The mutant enzymes E408Q and E442Q are inactive, whereas the specific enzymatic activity of the mutants R488Q, R488A, and Y554F are similar to that of the wild-type enzyme. These data strongly suggest that E408, E442, Y528, and H507 are involved in the catalytic process of TRH-DE while E464 presumably represents the third zinc-coordinating residue and may be equivalent to E166 in thermolysin. In contrast, amino acid residues R488 and Y554 seem not to be involved in the catalytic mechanism of TRH-DE.