Evidence by Site-Directed Mutagenesis That Arginine 203 of Thermolysin and Arginine 717 of Neprilysin (Neutral Endopeptidase) Play Equivalent Critical Roles in Substrate Hydrolysis and Inhibitor Bindi

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• 作者：Cynthia Marie-Claire ; Emmanuel Ruffet ; Serge Antonczak ; Ann Beaumont ; Michael O'Donohue ; Bernard P. Roques ; and Marie-Claude Fournié ; -Zaluski
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：November 11, 1997
• 年：1997
• 卷：36
• 期：45
• 页码：13938 - 13945
• 全文大小：171K
• 年卷期：v.36,no.45(November 11, 1997)
• ISSN：1520-4995

文摘

Neprilysin (neutral endopeptidase-24.11, EC 3.4.24.11) is amammalian zinc-endopeptidaseinvolved in the degradation of biologically active peptides.Although no atomic structure is available forthis enzyme, site-directed mutagenesis studies have shown that itsactive site resembles closely that ofthe bacterial zinc-endopeptidase, thermolysin (EC 3.4.24.27). Oneactive site residue of thermolysin,Arg-203, is involved in inhibitor binding by forming hydrogen bondswith the carbonyl group of a residuein the P₁' position and also participates in a hydrogenbond network involving Asp-170. Sequence alignmentdata shows that Arg-717 of neprilysin could play a similar role toArg-203 of thermolysin. This wasinvestigated by site-directed mutagenesis with Arg-203 of thermolysinand Arg-717 of neprilysin beingreplaced by methionine residues. This led, in both cases, todecreases in k_cat/K_mvalues, of 122-fold forneprilysin and 2300-fold for thermolysin, essentially due to changes ink_cat. The K_i valuesof severalinhibitors were also increased for the mutated enzymes. Inaddition, the replacement of Asp-170 ofthermolysin by Ala residue resulted in a decrease ink_cat/K_m of 220-fold.The results, coupled with amolecular modeling study, suggest that Arg-717 of neprilysincorresponds to Arg-203 of thermolysin andthat in both enzymes a hydrogen bond network exists, involving His-142,Asp-170, and Arg-203 inthermolysin and His-583, Asp-650, and Arg-717 in neprilysin, which iscrucial for hydrolytic activity.