Renirie R, Hemrika W, Piersma S R, Wever R
E.C. Slater Institute, Biocentrum Amsterdam, University of Amsterdam, Plantage Muidergracht 12, 1018 TV Amsterdam, The Netherlands.
Biochemistry. 2000 Feb 8;39(5):1133-41. doi: 10.1021/bi9921790.
The vanadate cofactor in vanadium chloroperoxidase has been studied using UV-VIS absorption spectroscopy. A band is present in the near-UV that is red-shifted as compared to free vanadate and shifts in both position and intensity upon change in pH. Mutation of vanadate binding residues has a clear effect on the spectrum. Substrate-induced spectral effects allow direct measurement of separate kinetics steps for the first time for vanadium haloperoxidases. A peroxo intermediate is formed upon addition of H(2)O(2), which causes a decrease in the absorption spectrum at 315 nm, as well as an increase at 384 nm. This peroxo form is very stable at pH 8.3, whereas it is less stable at pH 5.0, which is the optimal pH for activity. Upon addition of halides to the peroxo form, the native spectrum is re-formed as a result of halide oxidation. Stopped-flow experiments show that H(2)O(2) binding and Cl(-) oxidation occur on the millisecond to second time scale. These data suggest that the oxidation of Cl(-) to HOCl occurs in at least two steps. In the presence of H(2)O(2), the affinity for the vanadate cofactor was found to be much higher than previously reported for vanadate in the absence of H(2)O(2). This is attributed to the uptake of pervanadate by the apo-enzyme. Human glucose-6-phosphatase, which is evolutionarily related to vanadium chloroperoxidase, is also likely to have a higher affinity for pervanadate than vanadate. This could explain the enhanced insulin mimetic effect of pervanadate as compared to vanadate.
利用紫外可见吸收光谱法对钒氯过氧化物酶中的钒酸盐辅因子进行了研究。在近紫外区存在一个吸收带，与游离钒酸盐相比发生了红移，并且在pH值变化时其位置和强度都会发生改变。钒酸盐结合残基的突变对光谱有明显影响。底物诱导的光谱效应首次使得能够直接测量钒卤过氧化物酶的各个动力学步骤。加入H₂O₂后会形成过氧中间体，这会导致315 nm处的吸收光谱降低，同时384 nm处的吸收光谱增加。这种过氧形式在pH 8.3时非常稳定，而在pH 5.0（活性最佳pH）时稳定性较差。向过氧形式中加入卤化物后，由于卤化物氧化，会重新形成天然光谱。停流实验表明，H₂O₂结合和Cl⁻氧化发生在毫秒到秒的时间尺度上。这些数据表明，Cl⁻氧化为HOCl至少分两步进行。在存在H₂O₂的情况下，发现对钒酸盐辅因子的亲和力比之前报道的在不存在H₂O₂时钒酸盐的亲和力要高得多。这归因于脱辅基酶对过氧钒酸盐的摄取。与钒氯过氧化物酶在进化上相关的人葡萄糖-6-磷酸酶，可能对过氧钒酸盐的亲和力也比对钒酸盐的亲和力更高。这可以解释过氧钒酸盐与钒酸盐相比增强的胰岛素模拟效应。
