Burner U, Jantschko W, Obinger C
Institute of Chemistry, University of Agricultural Sciences, Vienna, Austria.
FEBS Lett. 1999 Jan 29;443(3):290-6. doi: 10.1016/s0014-5793(98)01727-x.
Myeloperoxidase (MPO) is the most abundant protein in neutrophils and plays a central role in microbial killing and inflammatory tissue damage. Because most of the non-steroidal anti-inflammatory drugs and other drugs contain a thiol group, it is necessary to understand how these substrates are oxidized by MPO. We have performed transient kinetic measurements to study the oxidation of 14 aliphatic and aromatic mono- and dithiols by the MPO intermediates, Compound I (k3) and Compound II (k4), using sequential mixing stopped-flow techniques. The one-electron reduction of Compound I by aromatic thiols (e.g. methimidazole, 2-mercaptopurine and 6-mercaptopurine) varied by less than a factor of seven (between 1.39 +/- 0.12 x 10(5) M(-1) s(-1) and 9.16 +/- 1.63 x 10(5) M(-1) s(-1)), whereas reduction by aliphatic thiols was demonstrated to depend on their overall net charge and hydrophobic character and not on the percentage of thiol deprotonation or redox potential. Cysteamine, cysteine methyl ester, cysteine ethyl ester and alpha-lipoic acid showed k3 values comparable to aromatic thiols, whereas a free carboxy group (e.g. cysteine, N-acetylcysteine, glutathione) diminished k3 dramatically. The one-electron reduction of Compound II was far more constrained by the nature of the substrate. Reduction by methimidazole, 2-mercaptopurine and 6-mercaptopurine showed second-order rate constants (k4) of 1.33 +/- 0.08 x 10(5) M(-1) s(-1), 5.25 +/- 0.07 x 10(5) M(-1) s(-1) and 3.03 +/- 0.07 x 10(3) M(-1) s(-1). Even at high concentrations cysteine, penicillamine and glutathione could not reduce Compound II, whereas cysteamine (4.27 +/- 0.05 x 10(3) M(-1) s(-1)), cysteine methyl ester (8.14 +/- 0.08 x 10(3) M(-1) s(-1)), cysteine ethyl ester (3.76 +/- 0.17 x 10(3) M(-1) s(-1)) and alpha-lipoic acid (4.78 +/- 0.07 x 10(4) M(-1) s(-1)) were demonstrated to reduce Compound II and thus could be expected to be oxidized by MPO without co-substrates.
髓过氧化物酶（MPO）是中性粒细胞中含量最丰富的蛋白质，在杀灭微生物和炎症组织损伤中起核心作用。由于大多数非甾体抗炎药和其他药物都含有巯基，因此有必要了解这些底物是如何被MPO氧化的。我们采用连续混合停流技术进行了瞬态动力学测量，以研究MPO中间体化合物I（k3）和化合物II（k4）对14种脂肪族和芳香族单硫醇和二硫醇的氧化作用。芳香族硫醇（如甲硫咪唑、2-巯基嘌呤和6-巯基嘌呤）对化合物I的单电子还原作用变化小于7倍（在1.39±0.12×10⁵ M⁻¹ s⁻¹和9.16±1.63×10⁵ M⁻¹ s⁻¹之间），而脂肪族硫醇的还原作用则取决于其总体净电荷和疏水特性，而非硫醇去质子化的百分比或氧化还原电位。半胱胺、半胱氨酸甲酯、半胱氨酸乙酯和α-硫辛酸的k3值与芳香族硫醇相当，而游离羧基（如半胱氨酸、N-乙酰半胱氨酸、谷胱甘肽）则显著降低k3值。化合物II的单电子还原作用受底物性质的限制更大。甲硫咪唑、2-巯基嘌呤和6-巯基嘌呤的还原作用显示二级速率常数（k4）分别为1.33±0.08×10⁵ M⁻¹ s⁻¹、5.25±0.07×10⁵ M⁻¹ s⁻¹和3.03±0.07×10³ M⁻¹ s⁻¹。即使在高浓度下，半胱氨酸、青霉胺和谷胱甘肽也不能还原化合物II，而半胱胺（4.27±0.05×10³ M⁻¹ s⁻¹）、半胱氨酸甲酯（8.14±0.08×10³ M⁻¹ s⁻¹）、半胱氨酸乙酯（3.76±0.17×10³ M⁻¹ s⁻¹）和α-硫辛酸（4.78±0.07×10⁴ M⁻¹ s⁻¹）被证明可以还原化合物II，因此预计在没有共底物的情况下会被MPO氧化。
