Marquez L A, Dunford H B
Department of Chemistry, University of Alberta, Edmonton, Canada.
Arch Biochem Biophys. 1993 Sep;305(2):414-20. doi: 10.1006/abbi.1993.1440.
The myeloperoxidase-H2O2 system constitutes an effective physiological defense mechanism because of its ability to oxidize Cl- to HOCl, a powerful oxidizing agent, via the enzyme intermediate compound I. Two other oxidized intermediates, compounds II and III, are inactive in generation of HOCl. Acetaminophen, a safe drug at therapeutic doses but toxic at higher doses, was found to react with the oxidized intermediates of myeloperoxidase. Using steady-state kinetics on the chlorination of monochlorodimedon to measure the activity of myeloperoxidase, our study reveals that acetaminophen stimulates the chlorinating activity of the enzyme and optimum stimulation is achieved at about 30 microM. Increasing the concentration further causes a decline in the chlorination rate. The increase in enzyme activity at lower acetaminophen concentrations is accounted for by the increased turnover of compounds II and III to native enzyme, while the decline at higher acetaminophen concentrations is explained by the competition of acetaminophen with Cl- for compound I. Rapid scan and transient state kinetic results on the reaction of compound II and acetaminophen show that: (i) compound II does not pass through any other intermediate when acetaminophen reduces it back to native enzyme; and (ii) a simple binding interaction before enzyme reduction is involved. An apparent dissociation constant of 1.3 +/- 0.3 x 10(-4) M and a first-order rate constant for reduction of 37 +/- 4 s-1 were determined at 25 degrees C. Conventional spectral scans of the reaction between compound III and acetaminophen indicate that compound III goes back to native enzyme without any detectable intermediate. The rate of this reaction levels off at higher acetaminophen concentration. Rapid scans reveal that the reduction of compound I to compound II is faster in the presence of acetaminophen. Since the therapeutic concentrations of acetaminophen in man range approximately from 50 to 150 microM, the results of this study indicate that stimulation of myeloperoxidase activity is achieved within the safe dosage of the drug.
髓过氧化物酶-H₂O₂系统是一种有效的生理防御机制，因为它能够通过酶中间化合物I将Cl⁻氧化为次氯酸（一种强氧化剂）。另外两种氧化中间体，即化合物II和化合物III，在次氯酸生成过程中无活性。对乙酰氨基酚在治疗剂量下是一种安全药物，但在高剂量时有毒，它被发现能与髓过氧化物酶的氧化中间体发生反应。利用单氯二甲基酮氯化反应的稳态动力学来测量髓过氧化物酶的活性，我们的研究表明，对乙酰氨基酚能刺激该酶的氯化活性，在约30微摩尔时可实现最佳刺激效果。进一步增加浓度会导致氯化速率下降。较低对乙酰氨基酚浓度下酶活性的增加是由于化合物II和化合物III向天然酶的周转增加，而较高对乙酰氨基酚浓度下活性的下降是由于对乙酰氨基酚与Cl⁻竞争化合物I所致。关于化合物II与对乙酰氨基酚反应的快速扫描和瞬态动力学结果表明：（i）当对乙酰氨基酚将化合物II还原回天然酶时，化合物II不经过任何其他中间体；（ii）在酶还原之前涉及一种简单的结合相互作用。在25℃下测定的表观解离常数为1.3±0.3×10⁻⁴ M，还原的一级速率常数为37±4 s⁻¹。化合物III与对乙酰氨基酚反应的常规光谱扫描表明，化合物III回到天然酶时没有任何可检测到的中间体。该反应速率在较高对乙酰氨基酚浓度下趋于平稳。快速扫描显示，在对乙酰氨基酚存在下，化合物I还原为化合物II的速度更快。由于人体中对乙酰氨基酚的治疗浓度约为50至15微摩尔，本研究结果表明在该药物的安全剂量范围内可实现髓过氧化物酶活性的刺激。
