Reaction of serine-glyoxylate aminotransferase with the alternative substrate ketomalonate indicates rate-limiting protonation of a quinonoid intermediate
Reaction of serine-glyoxylate aminotransferase with the alternative substrate ketomalonate indicates rate-limiting protonation of a quinonoid intermediate
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DOI：
10.1021/bi051407p
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发表时间：
2005-12-06
期刊：
影响因子：
2.9
通讯作者：
Cook, PF
中科院分区：
文献类型：
--
作者：
Karsten, WE;Ohshiro, T;Cook, PF
丝氨酸-乙醛酸氨基转移酶（SGAT）是一种5 '-磷酸吡哆醛（PLP）酶，可催化L-丝氨酸和乙醛酸转化为羟基丙酮酸和甘氨酸。使用L-丝氨酸2-D的主要氘同位素效应是在稳态下对（V/K）（丝氨酸）和V的效应。前稳态实验还表明，没有主要的氘同位素效应与L-丝氨酸2-D。结果表明，在SGAT反应中不存在通过提取L-丝氨酸的α质子的速率限制。在稳定状态下，溶剂氘同位素效应约为2（V/K）（L-丝氨酸）和（V/K）（酮丙二酸）和约5.5 V。类似的溶剂同位素效应中观察到的预稳定状态的天然底物和替代底物酮丙二酸。在预稳定状态下，没有观察到典型的PLP酶的反应中间体与底物L-丝氨酸，乙醛酸，和羟基丙酮酸。这些数据表明，酮亚胺中间体的分解和形成是天然底物的主要限速步骤。相比之下，使用替代底物酮丙二酸酯，预稳态实验显示了典型的quinandine中间体的490 nm吸收物质的瞬态形成。溶剂同位素效应的结果还表明，酮丙二酸作为底物质子化在C-α是最慢的步骤中的SGAT反应。这是第一次报告的限速质子化的quinquinone在C-α的外部席夫碱在氨基转移酶反应。
Serine-glyoxylate aminotransferase (SGAT) from Hyphomicrobium methylovorum is a pyridoxal 5'-phosphate (PLP) enzyme that catalyzes the interconversion of L-serine and glyoxylate to hydroxypyruvate and glycine. The primary deuterium isotope effect using L-serine 2-D is one on (V/K)(serine) and V in the steady state. Pre-steady-state experiments also indicate that there is no primary deuterium isotope effect with L-serine 2-D. The results suggest there is no rate limitation by abstraction of the alpha proton of L-serine in the SGAT reaction. In the steady-state a solvent deuterium isotope effect of about 2 was measured on (V/K)(L-serine) and (V/K)(ketomalonate) and about 5.5 on V. Similar solvent isotope effects were observed in the pre-steady-state for the natural substrates and the alternative substrate ketomalonate. In the pre-steady-state, no reaction intermediates typical of PLP enzymes were observed with the substrates L-serine, glyoxylate, and hydroxypyruvate. The data suggest that breakdown and formation of the ketimine intermediate is the primary rate-limiting step with the natural substrates. In contrast, using the alternative substrate ketomalonate, pre-steady-state experiments display the transient formation of a 490 nm absorbing species typical of a quinonoid intermediate. The solvent isotope effect results also suggest that with ketomalonate as substrate protonation at C-alpha is the slowest step in the SGAT reaction. This is the first report of a rate-limiting protonation of a quinonoid at C-alpha of the external Schiff base in an aminotransferase reaction.