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Exploration of the S ′ 1 subsite of neprilysin: A joined molecular modeling and site‐directed mutagenesis study
Author(s) -
MarieClaire Cynthia,
Tiraboschi Gilles,
Ruffet Emmanuel,
Inguimbert Nicolas,
FournieZaluski MarieClaude,
Roques Bernard P.
Publication year - 2000
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/(sici)1097-0134(20000601)39:4<365::aid-prot90>3.0.co;2-t
Subject(s) - active site , site directed mutagenesis , mutagenesis , chemistry , stereochemistry , directed mutagenesis , molecular model , enzyme , binding site , docking (animal) , biochemistry , computational biology , mutation , biology , gene , mutant , medicine , nursing
Based on the recently described three‐dimensional model of the 507–749 region of neprilysin, which contains the catalytic site of the enzyme, experiments were performed to improve the proposed topology of its large and hydrophobic S ′ 1subsite. Docking studies, site‐directed mutagenesis, and biochemical studies were combined. The mutations of various residues proposed to be part of the S ′ 1subsite (F563A, F564A, M579A, F716A, and I718A) did not induce major structural reorganization of the active site as demonstrated by the slight modification of the enzyme activity. The mutations were also analyzed by measuring the inhibitory potencies of thiol inhibitors containing P ′ 1moieties of increasing sizes. These results combined with molecular modeling studies support the proposed topology of the S ′ 1subsite. This, and the critical role of F563 and M579 in inhibitor binding, could facilitate the synthesis of new potent and selective inhibitors. Proteins 2000;39:365–371. © 2000 Wiley‐Liss, Inc.