Pre-steady-state kinetic studies of mono- and binuclear metallo-beta-lactamases

J Spencer, AM Simm, CS Higgins, AR Clarke, TR Walsh

Research output: Contribution to journalArticle (Academic Journal)

Abstract

The metallo-beta-1actamases are a diverse group of Zn 2+ dependant hydrolases which share a zinc-binding HXHXD active site motif and the ability to hydrolyse third generation penicillin antibiotics (carbapenems). Although the subject of recent studies ~ understanding of the mechanism of these enzymes is incomplete, whilst the situation is further complicated by the existence of both mononuclear (single zinc) and binuclear (two zinc) enzymes with greatly differing substrate specificities. We report pre-steady state kinetic data on three metallo-beta-1actamases: 1. the specific carbapenemase ImiS from Aeromonas spp. which is mononuclear but appears to have a second inhibitory zinc-binding site 2. IMP-1 from Serratia marscescens, a binuclear enzyme turning over a broad range of beta-1actam substrates; 3. L1 from Stenotrophomonas maltophilia, a broad-spectrum binuclear enzyme that is tetrameric in solution. Hydrolysis of the colourimetric cephalosporin nitrocefm by L1 proceeds through a spectrally distinct anionic product-like intermediate previously suggested to be stabilised by interaction with Zn2 however similar species are not observed in reaction with either of the other two proteins. We have used protein fluorescence as an optical probe of events in hydrolysis of more physiologically relevant beta-1actam substrates by all three enzymes and conclude that, where populated, intermediates retain an intact beta-lactam bond and thus more closely resemble substrate. These results highlight the diversity of reaction mechanism existing within this group of proteins.
Translated title of the contributionPre-steady-state kinetic studies of mono- and binuclear metallo-beta-lactamases
Original languageEnglish
Pages (from-to)440 - 440
Number of pages1
JournalJournal of Inorganic Biochemistry
Volume86
Publication statusPublished - Aug 2001

Bibliographical note

Publisher: Elsevier
Name and Venue of Conference: 10th International Conference on Bioinorganic Chemistry, Florence, Italy
Conference Organiser: Bertini, I, Banci, L, Luchinat, C
Other: Abstracts Poster Presentations (Z7)

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