*Centre for Translational and Chemical Biology, School of Biological Sciences, University of Edinburgh, Michael Swann Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, U.K.
†Edinburgh Biomolecular NMR Unit, Joseph Black Building, University of Edinburgh, West Mains Road, Edinburgh, EH9 3JJ, U.K.
Biochem J. 2014 Mar 1;458(2):301-11. doi: 10.1042/BJ20130790.
The phosphotransfer mechanism of PYKs (pyruvate kinases) has been studied in detail, but the mechanism of the intrinsic decarboxylase reaction catalysed by PYKs is still unknown. 1H NMR was used in the present study to follow OAA (oxaloacetate) decarboxylation by trypanosomatid and human PYKs confirming that the decarboxylase activity is conserved across distantly related species. Crystal structures of TbPYK (Trypanosoma brucei PYK) complexed with the product of the decarboxylase reaction (pyruvate), and a series of substrate analogues (D-malate, 2-oxoglutarate and oxalate) show that the OAA analogues bind to the kinase active site with similar binding modes, confirming that both decarboxylase and kinase activities share a common site for substrate binding and catalysis. Decarboxylation of OAA as monitored by NMR for TbPYK has a relatively low turnover with values of 0.86 s-1 and 1.47 s-1 in the absence and presence of F26BP (fructose 2,6-bisphosphate) respectively. Human M1PYK (M1 isoform of PYK) has a measured turnover value of 0.50 s-1. The X-ray structures explain why the decarboxylation activity is specific for OAA and is not general for α-oxo acid analogues. Conservation of the decarboxylase reaction across divergent species is a consequence of piggybacking on the conserved kinase mechanism which requires a stabilized enol intermediate.
PYKs（丙酮酸激酶）的磷酸转移机制已被详细研究，但 PYKs 催化的内在脱羧反应机制仍不清楚。本研究采用 1H NMR 跟踪根足虫和人 PYKs 中的 OAA（草酰乙酸）脱羧作用，证实脱羧酶活性在亲缘关系较远的物种中是保守的。与脱羧反应产物（丙酮酸）以及一系列底物类似物（D-苹果酸、2-酮戊二酸和草酸盐）复合的 TbPYK（布氏锥虫 PYK）晶体结构表明，OAA 类似物与激酶活性位点的结合模式相似，证实脱羧酶和激酶活性都共享一个共同的底物结合和催化位点。通过 NMR 监测 TbPYK 的 OAA 脱羧作用，其周转率相对较低，分别为 0.86 s-1 和 1.47 s-1，在不存在和存在 F26BP（果糖 2,6-双磷酸）的情况下。人 M1PYK（PYK 的 M1 同工型）的测量周转率值为 0.50 s-1。X 射线结构解释了为什么脱羧酶活性特异性针对 OAA，而不是针对α-酮酸类似物的一般性。不同物种之间脱羧反应的保守性是其依赖于保守激酶机制的结果，该机制需要稳定的烯醇中间物。
