Unusual mechanism of oxygen atom transfer and product rearrangement in the catalytic reaction of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase
Unusual mechanism of oxygen atom transfer and product rearrangement in the catalytic reaction of 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase
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DOI：
10.1021/bi970089u
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发表时间：
1997-07-01
期刊：
影响因子：
2.9
通讯作者：
Massey, V
中科院分区：
文献类型：
--
作者：
Chaiyen, P;Brissette, P;Massey, V
研究了 2-甲基-3-羟基吡啶-5-羧酸 (MHPC) 加氧酶与底物 MHPC 的氧化反应。发现了两种氧化黄素中间体 C(4a)-氢过氧黄素和 C(4a)-羟基黄素，这意味着该酶的功能与黄素蛋白羟化酶类似。这一发现得到了独立氧 18 示踪剂实验结果的支持，该实验表明 O-18(2) 中的一个氧原子和 (H2O)-O-18 中的一个氧原子被纳入产物中。 MHPC 加氧酶通常催化 MHPC 吡啶环的氧化和水解开环，产生无环化合物 α-(N-乙酰氨基亚甲基)琥珀酸。使用不含 2-甲基的 5-羟基烟酸 (5HN)，我们测试了水解反应是否是由于 MHPC 上存在 2-甲基（阻止初始产物的重新芳构化）或 MHPC 加氧酶的特定性质所致。 5HN 和 MHPC 加氧酶的酶促反应的产物分析表明，该酶催化羟基化以及随后羟基化底物的水解，产生无环产物。对氧化反应的研究表明，该酶使用与 MHPC 反应相同的机制来催化 5HN 反应。
The oxygenation reaction of 2-methyl-3-hydroxypyridine-5-carboxylic acid (MHPC) oxygenase with the substrate, MHPC, was investigated. Two oxygenated flavin intermediates C(4a)-hydroperoxy flavin and C(4a)-hydroxy flavin were found, implying that the enzyme functions similarly to flavoprotein hydroxylases. This finding is supported by the results of independent oxygen-18 tracer experiments, which showed that one atom of oxygen from O-18(2) and one atom of oxygen from (H2O)-O-18 are incorporated in the product. MHPC oxygenase normally catalyzes both the oxygenation and the hydrolytic ring opening of the pyridine ring of MHPC to yield the acyclic compound, alpha-(N-acetylaminomethylene)succinic acid. Using 5-hydroxynicotinic acid (5HN), which has no 2-methyl group, we tested whether the hydrolytic reaction was due to the presence of the 2-methyl group on MHPC (that prevented rearomatization of the initial product) or to the specific properties of MHPC oxygenase. Product analysis of the enzymatic reaction of 5HN and MHPC oxygenase shows that the enzyme catalyzes the hydroxylation and subsequent hydrolysis of the hydroxylated substrate to yield an acyclic product. The investigation of the oxygenation reaction demonstrates that the enzyme uses the same mechanism to catalyze the 5HN reaction as it does in the MHPC reaction.