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首页> 外文期刊>Biochemistry >Reaction of 2-methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase with N-methyl-5-hydroxynicotinic acid: studies on the mode of binding, and protonation status of the substrate
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Reaction of 2-methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase with N-methyl-5-hydroxynicotinic acid: studies on the mode of binding, and protonation status of the substrate

机译：2-甲基-3-羟基吡啶-5-羧酸（MHPC）加氧酶与N-甲基-5-羟基烟酸的反应：底物的结合方式和质子化状态研究

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摘要

Titrations of 2-methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase with the substrate MHPC identified the MHPC species bound to the enzyme as the tripolar ionic species. This result was supported by studies of the binding to the enzyme of N-methyl-5-hydroxynicotinic acid (NMHN), an MHPC analog existing only in the tripolar ionic form. The Kd is 55 microM compared to a Kd of 9.2 microM for MHPC and 5.2 microM for 5-hydroxynicotinic acid. Kinetics studies of the binding of NMHN to MHPC oxygenase show that its binding, like that for MHPC and for 5HN, is also a two-step process. Since NMHN never exists as an anionic form, neither of the observed steps is due to the binding of an anionic species as an intermediate step. Investigations of the reduction and oxygenation half reactions demonstrate that the mechanism of catalysis with NMHN is basically the same as with MHPC or with 5-hydroxynicotinic acid. Product analysis from reactions using NMHN, a compound that possesses positive charge on the nitrogen atom, indicates that the product of NMHN is an aliphatic compound, similar to the products derived from MHPC and from another substrate analog, 5-hydroxynicotinic acid. These results indicate that the nitrogen atom of the substrate is invariably protonated during the catalytic reaction.
机译：用底物MHPC滴定2-甲基-3-羟基吡啶-5-羧酸（MHPC）加氧酶的过程将与该酶结合的MHPC物种识别为三极离子物种。 N-甲基-5-羟基烟酸（NMHN）（仅以三极离子形式存在的一种MHPC类似物）与酶的结合研究为该结果提供了支持。 Kd为55 microM，而MHPC的Kd为9.2 microM，5-羟基烟酸的Kd为5.2 microM。 NMHN与MHPC加氧酶结合的动力学研究表明，与MHPC和5HN结合一样，其结合也是一个两步过程。由于NMHN从未以阴离子形式存在，因此没有观察到的步骤归因于作为中间步骤的阴离子种类的结合。对还原和氧合半反应的研究表明，用NMHN催化的机理与用MHPC或5-羟基烟酸基本相同。使用NMHN（一种在氮原子上带正电荷的化合物）进行的反应进行的产物分析表明，NMHN的产物是一种脂肪族化合物，类似于MHPC和另一种底物类似物5-羟基烟酸的产物。这些结果表明，在催化反应期间，底物的氮原子始终被质子化。

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