Furtmüller P G, Obinger C, Hsuanyu Y, Dunford H B
Institute of Chemistry, University of Agricultural Sciences, Vienna, Austria.
Eur J Biochem. 2000 Oct;267(19):5858-64. doi: 10.1046/j.1432-1327.2000.01491.x.
The reaction of myeloperoxidase compound I (MPO-I) with chloride ion is widely assumed to produce the bacterial killing agent after phagocytosis. Two values of the rate constant for this important reaction have been published previously: 4.7 x 106 M-1.s-1 measured at 25 degrees C [Marquez, L.A. and Dunford, H.B. (1995) J. Biol. Chem. 270, 30434-30440], and 2.5 x 104 M-1.s-1 at 15 degrees C [Furtmüller, P.G., Burner, U. & Obinger, C. (1998) Biochemistry 37, 17923-17930]. The present paper is the result of a collaboration of the two groups to resolve the discrepancy in the rate constants. It was found that the rate constant for the reaction of compound I, generated from myeloperoxidase (MPO) and excess hydrogen peroxide with chloride, decreased with increasing chloride concentration. The rate constant published in 1995 was measured over a lower chloride concentration range; the 1998 rate constant at a higher range. Therefore the observed conversion of compound I to native enzyme in the presence of hydrogen peroxide and chloride ion cannot be attributed solely to the single elementary reaction MPO-I + Cl- --> MPO + HOCl. The simplest mechanism for the overall reaction which fit the experimental data is the following: MPO+H2O2 ⇄k-1k1 MPO-I+H2O MPO-I+Cl- ⇄k-2k2 MPO-I-Cl- MPO-I-Cl- -->k3 MPO+HOCl where MPO-I-Cl- is a chlorinating intermediate. We can now say that the 1995 rate constant is k2 and the corresponding reaction is rate-controlling at low [Cl-]. At high [Cl-], the reaction with rate constant k3 is rate controlling. The 1998 rate constant for high [Cl-] is a composite rate constant, approximated by k2k3/k-2. Values of k1 and k-1 are known from the literature. Results of this study yielded k2 = 2.2 x 106 M-1.s-1, k-2 = 1.9 x 105 s-1 and k3 = 5.2 x 104 s-1. Essentially identical results were obtained using human myeloperoxidase and beef spleen myeloperoxidase.
髓过氧化物酶化合物I（MPO-I）与氯离子的反应被广泛认为在吞噬作用后产生细菌杀伤剂。此前已发表了该重要反应速率常数的两个值：在25℃下测得为4.7×10⁶ M⁻¹·s⁻¹[马尔克斯，L.A.和邓福德，H.B.（1995年）《生物化学杂志》270，30434 - 30440]，以及在15℃下为2.5×10⁴ M⁻¹·s⁻¹[富尔特米勒，P.G.，布尔纳，U.和奥宾格，C.（1998年）《生物化学》37，17923 - 17930]。本文是两组合作解决速率常数差异的结果。发现由髓过氧化物酶（MPO）和过量过氧化氢与氯离子生成的化合物I的反应速率常数随氯离子浓度增加而降低。1995年发表的速率常数是在较低氯离子浓度范围内测得的；1998年的速率常数是在较高浓度范围内测得的。因此，在过氧化氢和氯离子存在下观察到的化合物I向天然酶的转化不能仅归因于单一基元反应MPO-I + Cl⁻ → MPO + HOCl。符合实验数据的总反应最简单机制如下：
MPO + H₂O₂ ⇄ k₁/k₋₁ MPO-I + H₂O
MPO-I + Cl⁻ ⇄ k₂/k₋₂ MPO-I-Cl⁻
MPO-I-Cl⁻ → k₃ MPO + HOCl
其中MPO-I-Cl⁻是氯化中间体。我们现在可以说1995年的速率常数是k₂，相应反应在低[Cl⁻]时是速率控制步骤。在高[Cl⁻]时，速率常数为k₃的反应是速率控制步骤。1998年高[Cl⁻]时的速率常数是一个复合速率常数，近似为k₂k₃/k₋₂。k₁和k₋₁的值可从文献中得知。本研究结果得出k₂ = 2.2×10⁶ M⁻¹·s⁻¹，k₋₂ = 1.9×10⁵ s⁻¹，k₃ = 5.2×10⁴ s⁻¹。使用人髓过氧化物酶和牛脾髓过氧化物酶获得了基本相同的结果。
