Exploration of the catalytic site of endopeptidase 24.11 by site‐directed mutagenesis Histidine residues 583 and 587 are essential for catalysis

FEBS Letters

Sales, Valérie¹  Nault, Christiane¹  Boileau, Guy¹  Devault, Alain¹  Beaumont, Ann²  Roques, Bernard²  Crine, Philippe¹ 

[1] Département de biochimie, Université de Montréal, Montréal, Canada;Département de Chimie Organique, U 266 Institut de la Santé et de la Recherche Médicale, UA 498 Centre National de la Recherche Scientifique, Faculté de Pharmacie, Paris, France

关键词: Endopeptidase 24.11;    Histidine;    Catalytic site;    Site-directed mutagenesis;   

DOI  :  10.1016/0014-5793(88)80701-4

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown