EXPLORATION OF THE CATALYTIC SITE OF ENDOPEPTIDASE 24.11 BY SITE-DIRECTED MUTAGENESIS - HISTIDINE RESIDUE-583 AND RESIDUE-587 ARE ESSENTIAL FOR CATALYSIS
EXPLORATION OF THE CATALYTIC SITE OF ENDOPEPTIDASE 24.11 BY SITE-DIRECTED MUTAGENESIS - HISTIDINE RESIDUE-583 AND RESIDUE-587 ARE ESSENTIAL FOR CATALYSIS
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DOI：
10.1016/0014-5793(88)80701-4
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发表时间：
1988-04-11
期刊：
影响因子：
3.5
通讯作者：
BOILEAU, G
中科院分区：
文献类型：
--
作者：
DEVAULT, A;SALES, V;BOILEAU, G
中性内肽酶（NEP，EC 3.4.24.11）与嗜热菌蛋白酶（一种具有相似特异性的细菌金属内肽酶）的一级结构的直接比较显示，除了两个保守的短片段之外，两个序列之间几乎没有相似性。在嗜热菌蛋白酶中，这些片段含有几个参与催化的残基，包括两个锌配位组氨酸（His-142和His-146）和第三个组氨酸（His-231），通过氢键稳定过渡态。通过定点突变NEP cDNA并在COS-1细胞中表达，探讨了NEP中相应组氨酸（His-583、His-587和His-637）的作用。NEP的His-583或His-587取代Phe完全消除了重组酶的活性和锌定向抑制剂识别，表明这些残基作为锌配体发挥类似于嗜热菌蛋白酶的His- 142和His-146的作用。相比之下，His-637取代苯丙氨酸残基对酶活性没有影响。
Direct comparison of the primary structure of neutral endopeptidase (NEP, EC 3.4.24.11) with that of thermolysin, a bacterial metalloendopeptidase with a similar specificity, has revealed very few similarities between the two sequences, except for two conserved short segments. In thermolysin, these segments contain several of the residues involved in catalysis, including two zinc coordinating histidines (His-142 and His-146) and a third histidine (His-231) involved in stabilizing the transition state through hydrogen bonding. The role of the corresponding histidines in NEP (His-583, His-587 and His-637) was explored by site-directed mutagenesis of NEP cDNA and expression of the mutated cDNA in COS-1 cells. Substitution of either His-583 or His-587 of NEP for Phe completely abolished the activity and Zn-directed inhibitor recognition of the recombinant enzyme, suggesting that these residues play a role similar to His- 142 and His-146 of thermolysin as zinc ligands. In contrast, substitution of His-637 for a phenylalanine residue was without effect on enzyme activity.