当前位置： SCI文献检索 > BIOCHEMISTRY期刊下所有文献 > Mechanism of inhibition of the class C beta-lactamase of Enterobacter cloacae P99 by phosphonate monoesters.

Mechanism of inhibition of the class C beta-lactamase of Enterobacter cloacae P99 by phosphonate monoesters.

Abstract：

:The class C serine beta-lactamase of Enterobacter cloacae P99 was inhibited by a series of aryl methylphosphonate monoester monoanions. The effectiveness of these inhibitors was promoted by an acylamido substituent on the methyl group and a good leaving group at phosphorus. The former preference suggests that noncovalent interaction of these inhibitors with the enzyme resembles that of substrates, while the latter suggests that nucleophilic displacement at phosphorus occurs as part of the inhibition mechanism. The truth of the latter proposition was confirmed by observation of release of 1 equiv of phenol concomitant with inhibition and of the presence of an equivalent amount of 14C-label on the enzyme after inhibition by a 14C-labeled phosphonate. The hydrolytically inert nature of the enzyme-inhibitor adduct, and its 31P chemical shift, suggested that O-phosphonylation of the enzyme had occurred. Although, by analogy with substrates, one might expect that the hydroxyl of the active site serine residue would be covalently modified by these inhibitors, successive alkali and acid treatment of the enzyme-inhibitor adduct generated no pyruvate. Instead, 1 equiv of lysinoalanine was found. This product was rationalized to arise through intramolecular capture by an adjacent lysine amine group of the dehydroalanine residue produced by alkali treatment of an O-phosphonylated serine residue. One equivalent of lysinoalanine was also produced by alkali treatment of the enzyme that had been inhibited by 6 beta-bromopenicillanic acid, a mechanism-based inhibitor known to acylate the hydroxyl group of the active site serine residue. It is therefore likely that the aryl phosphonates phosphonylate this residue. These compounds should be useful as beta-lactamase active site titrants and as sources of fresh insight into the chemical properties of the active site. The significant mechanistic features of the inhibition, in particular its strong leaving group dependence and the distinctive ability of the beta-lactamase active site to stabilize a dianionic transition state containing a pentacoordinated phosphorus, are discussed with respect to the active site structure. The comparison with phosph(or/on)yl inhibitors of serine proteinases is made, and the mechanism-based features of inhibition of serine hydrolases by phosph(on)ates are noted.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Rahil J,Pratt RF

doi

10.1021/bi00140a024

subject

Has Abstract

pub_date

1992-06-30 00:00:00

pages

5869-78

issue

eissn

0006-2960

issn

1520-4995

journal_volume

pub_type

杂志文章

在线工具

Mechanism of inhibition of the class C beta-lactamase of Enterobacter cloacae P99 by phosphonate monoesters.