Cryoenzymology of Bacillus cereus .beta.-lactamase II

Abstract

The effects of cryosolvents and subzero temperatures on the metalloenzyme beta-lactamase II from Bacillus cereus have been investigated. Preliminary experiments led to the selection of suitable systems for the study of beta-lactamase II catalysis at low temperatures, namely, cobalt(II) beta-lactamase II hydrolysis of benzylpenicillin in 60% (v/v) ethylene glycol and zinc beta-lactamase II hydrolysis of the chromophoric cephalosporin nitrocefin in 60% (v/v) methanol. Progress curves for the hydrolysis of benzylpenicillin by cobalt beta-lactamase II in 60% (v/v) ethylene glycol at temperatures below -30 degrees C consisted of a transient followed by a steady-state phase. The amplitude of the transient implied a burst whose magnitude was greater than the concentration of enzyme, and the proposed mechanism comprises a branched pathway. The kinetics for the simplest variants of such pathways have been worked out, and the rate constants (and activation parameters) for the individual steps have been determined. The spectrum of the enzyme changed during turnover: when benzylpenicillin was added to cobalt beta-lactamase II, there was a large increase in the cysteine-cobalt(II) charge-transfer absorbance at 333 nm. This increase occurred within the time of mixing, even at -50 degrees C. The subsequent decrease in A333 was characterized by a rate constant that had the same value as the "branching" rate constant of the branched-pathway mechanism. This step is believed to be a change in conformation of the enzyme-substrate complex. Single-turnover experiments utilized the change in A333, and the results were consistent with pre-steady-state and steady-state experiments. When a single-turnover experiment at -48 degrees C was quenched with acid, the low molecular weight component of the intermediate was shown to be substrate. The mechanism advanced for the hydrolysis of benzylpenicillin by cobalt beta-lactamase II involves two noncovalent enzyme-substrate complexes that have been characterized by their electronic absorption spectra. When manganese beta-lactamase II was used, the same features (implying a branched pathway) were evident; these experiments were carried out at ordinary temperatures and did not utilize a cryosolvent. The hydrolysis of nitrocefin by zinc beta-lactamase II has been studied concurrently in 60% (v/v) methanol. Progress curves were triphasic. There were two transients preceding the linear steady-state phase. The stoichiometry of the burst again implied a branched pathway.(ABSTRACT TRUNCATED AT 400 WORDS)

Metal Ion Binding and Coordination Geometry for Wild Type and Mutants of Metallo-β-lactamase from Bacillus cereus569/H/9 (BcII)

“…The affinity for Co(II) is thus two orders of magnitude weaker than for Zn(II) because of the lower value for the association rate k on,1 for Co(II). The spectral features obtained at a [Co(II)]/[enzyme] ratio of 1 are identical to those described earlier (23). Increasing the [Co(II)]/[enzyme] ratio above 1 results in an additional charge transfer band at 383 nm.…”

“…The same spectral changes as described here were obtained in 15 mM sodium cacodylate, pH 7.0 (data not shown). Earlier studies were performed in succinate buffer at pH 6.3 (24) or in a cryosolvent containing 60% ethylene glycol in 80 mM sodium cacodylate, pH 6.4 (glass electrode reading in aqueous-organic solvent) (23). It might be assumed that solvent composition, the presence of succinate, and/or the lower pH are responsible for the different spectroscopic behavior.…”

Mechanistic study of the hydrolysis of nitrocefin mediated by B.cereus metallo-β-lactamase

“…Put together, these results are consistent with a red shift of the substrate spectrum upon binding to the enzyme. An intermediate in nitrocefin hydrolysis by BcII with a red shifted absorption maximum was already reported by Bicknell and Waley in cryosolvents at sub zero temperatures 14 , who also reported that spectral shifts of this kind occur to nitrocefin when lowering the solvent polarity. Subclass B1 metallo-β-lactamases have a flexible loop which closes upon substrate binding [15][16][17] .…”

Coordination Geometries of Metal Ions in D- or L-Captopril-inhibited Metallo-β-lactamases

“…It is likely that a replacement of water by sulfur in the H-site results in the observed changes (compare Ref. 29). The appearance of two additional bands at 310 and 375 nm can be attributed to sulfurcobalt ligand-to-metal charge transfer due to binding of the captopril sulfur.…”