Cryoenzymology of Bacillus cereus .beta.-lactamase II

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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.degree. 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.degree. 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.degree. 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. The kinetics for a mechanism in which there are three intermediates (two of them lying in the branch) have been worked out and used to obtain values (and activation parameters) for four of the rate constants. Single-turnover experiments confirmed the kinetic scheme. An intermediate was detected spectroscopically, its calculated absorption spectrum resembled that of the substrate shifted to longer wavelengths. Low-temperature chromatography, carried out at varying intervals of time, revealed a second intermediate, accumulating at the expected rate. Acid-quench experiments suggested that all three intermediates were noncovalent enzyme-substrate complexes. The observation of pre-steady-state kinetics requiring a branched catalytic pathway for the zinc, manganese(II), and cobalt(II) forms of .beta.-lactamase II provides convincing evidence for the proposed kinetic model. Branched pathways that comprise conformationally distinct complexes may be a consequence of protein fluctuations.