Multiple turnovers of the nicotino-enzyme PdxB require α-keto acids as cosubstrates.
Multiple turnovers of the nicotino-enzyme PdxB require α-keto acids as cosubstrates.
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烟碱 - 酶PDXB的多个失误需要α-酮酸作为cosubstrates。
DOI：
10.1021/bi101291d
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发表时间：
2010-11-02
期刊：
影响因子：
2.9
通讯作者：
Copley, Shelley D.
中科院分区：
文献类型：
--
作者：
Rudolph, Johannes;Kim, Juhan;Copley, Shelley D.
PdxB催化磷酸吡哆醛生物合成的第二步，将4-磷酸-D-阿糖胞苷（4PE）氧化为2-氧代-3-羟基-4-磷酸-丁酸（OHPB），同时将NAD+还原为NADH。PdxB是一种烟碱酶，其中NAD（H）辅因子保持与PdxB紧密结合。PdxB如何进行多次转换一直是个谜，因为在4PE转化为OHPB后，添加游离NAD+不会再氧化酶结合的NADH。我们已经解决了这个谜，证明了各种生理上可用的α-酮酸作为氧化剂的PdxB，以维持多次周转。在使用磷酸吡哆醛生物合成途径的下两种酶（SerC和PdxA）的偶联试验中，我们发现α-酮戊二酸、草酰乙酸和丙酮酸同样是PdxB的良好底物（kcat/Km值约为1 × 104 M-1 s-1）。底物4PE的动力学参数包括1.4 s-1的kcat、2.9 μM的Km和6.7 × 106 M-1 s-1的kcat/Km。此外，我们通过显示D-2-HGA（而非L-2-HGA）是与4PE相比的竞争性抑制剂和与α-酮戊二酸相比的非竞争性抑制剂，表征了α-酮戊二酸还原的立体化学。
PdxB catalyzes the second step in the biosynthesis of pyridoxal phosphate by oxidizing 4-phospho-D-erythronate (4PE) to 2-oxo-3-hydroxy-4-phospho-butanoate (OHPB) with concomitant reduction of NAD+ to NADH. PdxB is a nicotino-enzyme wherein the NAD(H) cofactor remains tightly bound to PdxB. It has been a mystery how PdxB performs multiple turnovers since addition of free NAD+ does not re-oxidize the enzyme-bound NADH following conversion of 4PE to OHPB. We have solved this mystery by demonstrating that a variety of physiologically available α-ketoacids serve as oxidants of PdxB to sustain multiple turnovers. In a coupled assay using the next two enzymes of the biosynthetic pathway for pyridoxal phosphate (SerC and PdxA), we have found that α-ketoglutarate, oxaloacetic acid, and pyruvate are equally good substrates for PdxB (kcat/Km values ~ 1 × 104 M-1s-1). The kinetic parameters for the substrate 4PE include a kcat of 1.4 s-1, a Km of 2.9 μM, and a kcat/Km of 6.7 × 106 M-1s-1. Additionally, we have characterized the stereochemistry of α-ketoglutarate reduction by showing that D-2-HGA, but not L-2-HGA, is a competitive inhibitor vs. 4PE and a noncompetitive inhibitor vs. α-ketoglutarate.
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影响因子：
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