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. 1987 Oct 1;247(1):29–33. doi: 10.1042/bj2470029

Inactivation of the thiol RTEM-1 beta-lactamase by 6-beta-bromopenicillanic acid. Identity of the primary active-site nucleophile.

A K Knap 1, R F Pratt 1
PMCID: PMC1148364  PMID: 2825657

Abstract

The thiol RTEM-1 beta-lactamase [Sigal, Harwood & Arentzen (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7157-7160] is inactivated by 6-beta-bromopenicillanic acid with formation of a characteristic chromophore, absorbing maximally at 350 nm, which is covalently bound to the enzyme. Model studies suggest that the chromophore is that of a 6-carboxylate thiol ester of 2,3-dihydro-2,2-dimethyl-1,4-thiazine-3,6-dicarboxylate, which can arise by rearrangement of the thiol-penicilloate obtained by thiolysis of the beta-lactam of 6-beta-bromopenicillanate. Loss of activity of the enzyme is also concerted with disappearance of its single (cysteine) thiol group. These results indicate that the thiol group of this enzyme is indeed a nucleophilic catalyst in beta-lactam turnover. The thiol beta-lactamase is also inactivated by clavulanic acid with formation of a chromophore, presumably a 3-aminoacrylate thiol ester, at 308 nm. Both 6-beta-bromopenicillanate and clavulanate are hydrolysed more slowly by the thiol enzyme than by the native serine beta-lactamase, but, probably as a consequence of this, both compounds inactivate the former enzyme more efficiently. Cefoxitin, a poor substrate of the native enzyme, does not appear to interact covalently with the thiol beta-lactamase.

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Selected References

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