DeLuca D C, Dennis R, Smith W G
University of Arkansas for Medical Sciences, Little Rock 72205, USA.
Arch Biochem Biophys. 1995 Jun 20;320(1):129-34. doi: 10.1006/abbi.1995.1350.
We have quantitatively compared the rates of peroxide-dependent inactivation of bovine liver catalase and Aspergillus niger catalase as class representatives of catalases that contain tightly bound NADPH and those that do not. Inactivation of these catalases in the presence of ethanol has also been quantitated in an effort to assess the importance of compound II, an inactive form of bovine liver catalase, in the inactivation reaction. The values of k2, the second-order rate constant for inactivation calculated for the bovine enzyme, in the absence and presence of ethanol, respectively, were 8.9 +/- 0.26 and 8.5 +/- 0.27 M-1 min-1. In contrast, the values for the A. niger enzyme were 0.51 +/- 0.069 and 10.5 +/- 0.32 M-1 min-1. The A. niger enzyme is more stable toward hydrogen peroxide-induced inactivation than the liver enzyme. The A. niger enzyme is markedly destabilized by 20 mM ethanol, whereas the inactivation of the liver enzyme is unaffected by ethanol. Reaction of bovine liver catalase with ethyl hydroperoxide produced the characteristic absorption spectrum of compound I and in the absence of ethanol the spectrum associated with compound II. In contrast, the fungal enzyme developed compound I spectrum but spectral changes that might be ascribed to compound II were barely detected in the Soret region. Spectral changes for A. niger catalase in the visible region were modified by the presence of ethanol but could not be clearly correlated with the bovine catalase compound II spectra either in the presence or absence of ethanol. The stability of the fungal and bovine catalases in the presence of hydrogen peroxide is quantitatively documented. The enzymes are also shown to be different in their response to ethanol and in the formation of compound II-like species with ethyl hydroperoxide. It appears unlikely that compound II is an intermediate in the hydrogen peroxide-mediated inactivation reaction of either catalase under catalatic assay conditions.
我们已对牛肝过氧化氢酶和黑曲霉过氧化氢酶的过氧化物依赖性失活速率进行了定量比较，它们分别作为含有紧密结合的NADPH的过氧化氢酶和不含紧密结合的NADPH的过氧化氢酶的类别代表。为了评估牛肝过氧化氢酶的无活性形式化合物II在失活反应中的重要性，我们还对这些过氧化氢酶在乙醇存在下的失活情况进行了定量。牛酶在不存在和存在乙醇时计算得到的失活二级速率常数k2值分别为8.9±0.26和8.5±0.27 M-1 min-1。相比之下，黑曲霉酶的值分别为0.51±0.069和10.5±0.32 M-1 min-1。黑曲霉酶对过氧化氢诱导的失活比肝酶更稳定。20 mM乙醇会使黑曲霉酶明显失稳，而肝酶的失活不受乙醇影响。牛肝过氧化氢酶与氢过氧化乙酯反应产生了化合物I的特征吸收光谱，在不存在乙醇的情况下产生了与化合物II相关的光谱。相比之下，真菌酶产生了化合物I光谱，但在索雷特区域几乎未检测到可能归因于化合物II的光谱变化。乙醇的存在改变了黑曲霉过氧化氢酶在可见光区域的光谱变化，但在存在或不存在乙醇的情况下，都无法与牛过氧化氢酶化合物II光谱明确关联。定量记录了真菌和牛过氧化氢酶在过氧化氢存在下的稳定性。还表明这两种酶对乙醇的反应以及与氢过氧化乙酯形成类似化合物II的物种有所不同。在催化测定条件下，化合物II似乎不太可能是任何一种过氧化氢酶的过氧化氢介导失活反应的中间体。
