SPECTRAL AND KINETIC-STUDIES ON THE FORMATION OF MYELOPEROXIDASE COMPOUND-I AND COMPOUND-II - ROLES OF HYDROGEN-PEROXIDE AND SUPEROXIDE
SPECTRAL AND KINETIC-STUDIES ON THE FORMATION OF MYELOPEROXIDASE COMPOUND-I AND COMPOUND-II - ROLES OF HYDROGEN-PEROXIDE AND SUPEROXIDE
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DOI：
10.1021/bi00172a022
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发表时间：
1994-02-15
期刊：
影响因子：
2.9
通讯作者：
DUNFORD, HB
中科院分区：
文献类型：
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作者：
MARQUEZ, LA;HUANG, JT;DUNFORD, HB
髓过氧化物酶在H2 O2的存在下，化合物I和II的转换已被重新研究，以解释化合物I形成的异常化学计量和化合物I到化合物II的快速自发衰变。快速扫描研究表明，至少需要20倍过量的H2 O2才能获得相对纯的化合物I的良好光谱; H2 O2浓度的进一步增加导致化合物I被还原为化合物II，这是一种非常稳定的中间体。化合物I的形成是可逆的，表观二级正向速率常数为（1.8 +/- 0.1）× 10（7）M（-1）s（-1），反向速率常数为58 +/- 4 s（-1），化合物I解离为天然酶和H2O的常数为3.2 μ M。这种可逆性是可以解释形成化合物I所需的大量过量H2 O2的一个因素。由化合物I和H2 O2形成化合物II的表观二级速率常数为（8.2 +/- 0.2）× 10（4）M（-1）s（-1）。我们确认pH依赖性研究，这表明，化合物I和II的形成是由酶中的一个残基与pK（a）约4.0控制。过量的H2 O2也通过酶的过氧化氢酶活性转化为O-2。然而，我们不认为这是一个主要途径，因为它不能解释化合物I快速自发还原为化合物II。化合物I的衰变和化合物II的形成的时间过程是双相的。根据化合物I还原为化合物II的两种可能途径解释了两相动力学：与H_2O_2的快速反应和与O-2（-）作为还原剂的缓慢反应。增加H_2O_2的浓度，降低pH值从中性到酸性值，或添加SOD到系统中的结果在两个阶段的收敛到一个两相过程。通过四硝基甲烷还原试验，在髓过氧化物酶/H2 O2系统中检测到超氧化物。还讨论了涉及酶制剂中的蛋白质部分和杂质的化合物I转化为化合物II的自还原途径的可能性。化合物II可能与产生的O-2（-）反应形成天然酶，但仅在非常有限的程度上。我们排除血红素不等价作为一个可能的解释异常化学计量的化合物I形成的氰化物结合研究的二聚酶和它的单体，这表明，两个血红素辅基的酶具有相同的反应性。
The conversion of myeloperoxidase to compounds I and II in the presence of H2O2 has been reinvestigated in order to explain the abnormal stoichiometry of compound I formation and the fast spontaneous decay of compound I to compound II. Rapid-scan studies show that at least a 20-fold excess of H2O2 is required to obtain a good spectrum of relatively pure compound I; a further increase in H2O2 concentration causes compound I to be reduced to compound II, which is a very stable intermediate. Compound I formation is reversible, with an apparent second-order forward rate constant of (1.8 +/- 0.1) x 10(7) M(-1) s(-1) and a reverse rate constant of 58 +/- 4 s(-1), giving a constant of 3.2 mu M for the dissociation of compound I to native enzyme and H2O. This reversibility is one factor that can explain the large excess of H2O2 required to form compound I. The apparent second-order rate constant for compound II formation from compound I and H2O2 is (8.2 +/- 0.2) x 10(4) M(-1) s(-1). We confirm pH dependence studies, which suggest that the formation of compounds I and II is controlled by a residue in the enzyme with a pK(a) of about 4.0. Excess H2O2 is also converted to O-2 via catalase activity of the enzyme. However, we do not consider this a dominant pathway because it fails to account for the fast spontaneous reduction of compound I to compound II. The time courses for both the decay of compound I and the formation of compound II are biphasic. Biphasic kinetics was explained in terms of two possible routes of the reduction of compound I to compound II: a fast reaction with H2O2 and a slow reaction with O-2(-) as reducing agent. Increasing the concentration of H2O2, lowering the pH from neutral to acidic values, or adding SOD to the system results in the convergence of the two phases to a monophasic process. Superoxide was detected as a product in the myeloperoxidase/H2O2 system by the tetranitromethane reduction test. The possibility of an autoreduction pathway for the conversion of compound I to compound II involving the protein moiety and impurities in enzyme preparation is also discussed. Compound II possibly reacts with generated O-2(-) to form native enzyme, but only to a very limited extent. We exclude heme inequivalence as a possible explanation for the abnormal stoichiometry of compound I formation from cyanide binding studies on the dimeric enzyme and its monomer, which indicate that the two heme prosthetic groups of the enzyme have the same reactivity.