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

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.

Active-site serine mutants of the Streptomyces albus G β-lactamase

“…The interaction was studied on the basis of the model proposed by Frere et al (1982), which was slightly modified to accommodate the results described above for the wild-type enzyme (Scheme 2). On incubation of the S70C f-lactamase (6 /M) with 1 mM-fl-iodopenicillanate, a new rather wide absorbance band appeared between 290 and 370 nm, which could be explained by the sum of an enzyme-bound dihydrothiazine chromophore absorbing around 345 nm (Knap & Pratt, 1987) and the free dihydrothiazine absorbing at 305 nm in more-or-less equimolar proportions. After 4 h the absorbance at 345 nm was stabilized and corresponded to a nearly stoichiometric binding of the inactivator, while that at 305 nm slowly continued to increase.…”

Evidence from a mutant β-lactamase for the mechanism of β-lactamase-catalysed depsipeptide aminolysis

“…This level of activity could be reduced to 0.0005% of the wild-type activity by treatment with 6,1-bromopenicillanate. Since this inactivation was not accompanied by any measurable increase in absorption between 320 and 330 nm (the position of the characteristic chromophore generated on inhibition of the TEM,-lactamase by 6,/-bromopenicillanate [24]), it is probable that most of the residual activity must have been associated with a small amount of a contaminating Ser-70 ,J-lactamase, possibly the wild-type enzyme. The remaining activity may be that of the Ser-70 -+ Gly mutant active site, although some non-specific activity is also possible.…”

“…The concentrate was dialysed against 25 mM-imidazole buffer, pH 7.4. Further purification involved chromatofocusing followed by Sephadex G-75 chromatography, as previously described [23,24]. The yield of purified protein, which was homogeneous on SDS/PAGE, was 4 mg.…”

Inactivation of the RTEM-1 cysteine β-lactamase by iodoacetate. The nature of active-site functional groups and comparisons with the native enzyme

“…Enzyme concentrations and activities against benzylpenicillin were routinely determined spectrophotometrically as previously described [10,11]. The buffers used for the pH-rate profiles were 0.5 M-sodium acetate (pH 4-5.5), 0.1 M-sodium phosphate (pH 6-8) and 0.05 M-Bicine/HCI (pH 8-9.5).…”

“…We have recently approached the question of the chemical properties of the functional groups of the class A fl-lactamase active site through employment of the RTEM-1 cysteine ,J-lactamase. This enzyme was created by site-specific mutagenesis Cys) of the native RTEM-/J-lactamase [9], and does employ its active-site thiol group as the primary nucleophile in catalysis [10]. It is a useful target for investigation because the active-site thiol group is not only a much better intrinsic probe of the active site than the serine hydroxy group of the native enzyme, but also a more versatile point of attachment of extrinsic probes.…”