Marquez L A, Dunford H B
Department of Chemistry, University of Alberta, Edmonton, Canada.
J Biol Chem. 1995 Dec 22;270(51):30434-40. doi: 10.1074/jbc.270.51.30434.
The oxidation of lipoproteins is considered to play a key role in atherogenesis, and tyrosyl radicals have been implicated in the oxidation reaction. Tyrosyl radicals are generated in a system containing myeloperoxidase, H2O2, and tyrosine, but details of this enzyme-catalyzed reaction have not been explored. We have performed transient spectral and kinetic measurements to study the oxidation of tyrosine by the myeloperoxidase intermediates, compounds I and II, using both sequential mixing and single-mixing stopped-flow techniques. The one-electron reduction of compound I to compound II by tyrosine has a second order rate constant of (7.7 +/- 0.1) x 10(5) M-1 s-1. Compound II is then reduced by tyrosine to native enzyme with a second order rate constant of (1.57 +/- 0.06) x 10(4) M-1 s-1. Our study further revealed that, compared with horseradish peroxidase, thyroid peroxidase, and lactoperoxidase, myeloperoxidase is the most efficient catalyst of tyrosine oxidation at physiological pH. The second order rate constant for the myeloperoxidase compound I reaction with tyrosine is comparable with that of its compound I reaction with chloride: (4.7 +/- 0.1) x 10(6) M-1 s-1. Thus, although chloride is considered the major myeloperoxidase substrate, tyrosine is able to compete effectively for compound I. Steady state inhibition studies demonstrate that chloride binds very weakly to the tyrosine binding site of the enzyme. Coupling of tyrosyl radicals yields dityrosine, a highly fluorescent stable compound that had been identified as a possible marker for lipoprotein oxidation. We present spectral and kinetic data showing that dityrosine is further oxidized by both myeloperoxidase compounds I and II. The second order rate constants we determined for dityrosine oxidation are (1.12 +/- 0.01) x 10(5) M-1 s-1 for compound I and (7.5 +/- 0.3) x 10(2) M-1 s-1 for compound II. Therefore, caution must be exercised when using dityrosine as a quantitative index of lipoprotein oxidation, particularly in the presence of myeloperoxidase and H2O2.
脂蛋白的氧化被认为在动脉粥样硬化形成过程中起关键作用，并且酪氨酰自由基与氧化反应有关。酪氨酰自由基在含有髓过氧化物酶、过氧化氢和酪氨酸的体系中产生，但这种酶催化反应的细节尚未被探究。我们使用连续混合和单混合停流技术进行了瞬态光谱和动力学测量，以研究髓过氧化物酶中间体化合物I和化合物II对酪氨酸的氧化作用。酪氨酸将化合物I单电子还原为化合物II的二级速率常数为(7.7±0.1)×10⁵ M⁻¹ s⁻¹。然后化合物II被酪氨酸还原为天然酶，二级速率常数为(1.57±0.06)×10⁴ M⁻¹ s⁻¹。我们的研究进一步表明，与辣根过氧化物酶、甲状腺过氧化物酶和乳过氧化物酶相比，髓过氧化物酶在生理pH值下是酪氨酸氧化最有效的催化剂。髓过氧化物酶化合物I与酪氨酸反应的二级速率常数与其化合物I与氯离子反应的二级速率常数相当：(4.7±0.1)×10⁶ M⁻¹ s⁻¹。因此，尽管氯离子被认为是髓过氧化物酶的主要底物，但酪氨酸能够有效地竞争化合物I。稳态抑制研究表明，氯离子与该酶的酪氨酸结合位点结合非常弱。酪氨酰自由基的偶联产生二酪氨酸，一种高度荧光稳定的化合物，已被确定为脂蛋白氧化的可能标志物。我们提供的光谱和动力学数据表明，二酪氨酸会被髓过氧化物酶化合物I和化合物II进一步氧化。我们测定的二酪氨酸氧化的二级速率常数，化合物I为(1.12±０.01)×10⁵ M⁻¹ s⁻¹，化合物II为(7.5±0.3)×10² M⁻¹ s⁻¹。因此，在将二酪氨酸用作脂蛋白氧化的定量指标时必须谨慎，尤其是在存在髓过氧化物酶和过氧化氢的情况下。
