CRYOENZYMOLOGY OF BACILLUS-CEREUS BETA-LACTAMASE-II
CRYOENZYMOLOGY OF BACILLUS-CEREUS BETA-LACTAMASE-II
复制标题
DOI：
10.1021/bi00345a021
复制
发表时间：
1985-01-01
期刊：
影响因子：
2.9
通讯作者：
WALEY, SG
中科院分区：
文献类型：
--
作者：
BICKNELL, R;WALEY, SG
低温溶剂和零度以下的温度对金属酶β的影响已经研究了来自蜡状芽孢杆菌内酰胺酶II。初步实验导致选择合适的系统用于研究β-内酰胺酶II在低温下催化，即钴（II）β-内酰胺酶II在60%（v/v）乙二醇和β-锌中水解苄青霉素内酰胺酶II在60%（v/v）甲醇中水解发色头孢菌素硝头孢菌素。钴β-水解苄青霉素的进展曲线内酰胺酶II在60%（v/v）乙二醇中，温度低于-30 ℃。C由瞬态和随后的稳态相组成。瞬态的幅度意味着其幅度大于酶浓度的爆发，并且所提出的机制包括分支途径。这种途径的最简单的变体的动力学已经制定出来，并已确定的各个步骤的速率常数（和活化参数）。酶的谱在周转过程中发生变化：当苄青霉素加入钴β-内酰胺酶II，在333 nm处的半胱氨酸-钴（II）电荷转移吸光度大幅增加。这种增加发生在混合时间内，甚至在-50 ℃。C.随后A333的减少的特征在于具有与分支途径机制的“分支”速率常数相同的值的速率常数。该步骤被认为是酶-底物复合物构象的变化。单次翻转实验利用了A333的变化，结果与稳态前和稳态实验一致。当在-48 ℃下进行单次翻转实验时，C用酸淬灭，中间体的低分子量组分被证明是底物。提出了钴β-水解苄青霉素的机理。内酰胺酶II涉及两种非共价酶-底物复合物，其特征在于它们的电子吸收光谱。当锰β-当使用内酰胺酶II时，相同的特征（意味着分支途径）是明显的;这些实验在常温下进行并且不使用冷冻溶剂。头孢硝肟被锌β-在60%（v/v）甲醇中同时研究了内酰胺酶II。进展曲线呈三相性。在线性稳态阶段之前有两个瞬态。爆发的化学计量学再次暗示了一个分支途径。动力学的机制，其中有三个中间体（其中两个躺在分支）已制定出，并用于获得值（和活化参数）的四个速率常数。单周转实验证实了动力学方案。通过光谱检测到中间体，其计算的吸收光谱类似于向较长波长移动的底物的吸收光谱。在不同的时间间隔进行的低温色谱法显示了第二种中间体，以预期的速度积累。酸淬灭实验表明，所有三个中间体的非共价酶底物复合物。观察到β-环戊二烯基甲酸的锌、锰（II）和钴（II）形式需要分支催化途径的预稳态动力学。内酰胺酶II为所提出的动力学模型提供了令人信服的证据。包含构象不同复合物的分支途径可能是蛋白质波动的结果。
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.