Marquez L A, Huang J T, Dunford H B
Department of Chemistry, University of Alberta, Edmonton, Canada.
Biochemistry. 1994 Feb 15;33(6):1447-54. doi: 10.1021/bi00172a022.
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 microM 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 pKa of about 4.0. Excess H2O2 is also converted to O2 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.(ABSTRACT TRUNCATED AT 250 WORDS)
为了解释化合物I形成的异常化学计量关系以及化合物I快速自发衰变为化合物II的现象，我们重新研究了髓过氧化物酶在过氧化氢存在下向化合物I和化合物II的转化过程。快速扫描研究表明，至少需要20倍过量的过氧化氢才能获得相对纯净的化合物I的良好光谱；过氧化氢浓度的进一步增加会导致化合物I还原为化合物II，化合物II是一种非常稳定的中间体。化合物I的形成是可逆的，其表观二级正向速率常数为(1.8±0.1)×10⁷ M⁻¹ s⁻¹，逆向速率常数为58±4 s⁻¹，化合物I解离为天然酶和水的常数为3.2 μM。这种可逆性是可以解释形成化合物I所需大量过量过氧化氢的一个因素。由化合物I和过氧化氢形成化合物II的表观二级速率常数为(8.2±0.2)×10⁴ M⁻¹ s⁻¹。我们证实了pH依赖性研究，该研究表明化合物I和化合物II的形成受酶中一个pKa约为4.0的残基控制。过量的过氧化氢也会通过酶的过氧化氢酶活性转化为氧气。然而，我们不认为这是主要途径，因为它无法解释化合物I快速自发还原为化合物II的现象。化合物I的衰变和化合物II的形成的时间进程都是双相的。（摘要截短于250字）
