Tetrahedral intermediates in thiamin diphosphate-dependent decarboxylations exist as a 1',4'-imino tautomeric form of the coenzyme, unlike the michaelis complex or the free coenzyme.
Tetrahedral intermediates in thiamin diphosphate-dependent decarboxylations exist as a 1',4'-imino tautomeric form of the coenzyme, unlike the michaelis complex or the free coenzyme.
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DOI：
10.1021/bi049549r
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发表时间：
2004-06
期刊：
影响因子：
2.9
通讯作者：
N. Nemeria;A. Baykal;Ebenezer Joseph;S. Zhang;Yan‐Ping Yan;W. Furey;F. Jordan
中科院分区：
文献类型：
--
作者：
N. Nemeria;A. Baykal;Ebenezer Joseph;S. Zhang;Yan‐Ping Yan;W. Furey;F. Jordan
在酵母丙酮酸脱羧酶（YPDC）和大肠杆菌丙酮酸脱氢酶复合物（PDHc-E1）的E1亚基上观察到二磷酸硫胺素（ThDP）依赖性酶的两个圆二色性信号，一个在300 -305 nm范围内的正带和一个在320-330 nm范围内的负带。将四面体ThDP-乙醛加合物2-α-羟乙基ThDP加入PDHc-E1中，在300 nm处产生正带，与1 ′，4 ′-亚氨基嘧啶互变异构体的形成一致，这也证明了膦酰基乙酰基硫胺素二磷酸，一种四面体ThDP-丙酮酸加合物2-α-乳酰基ThDP的稳定类似物（Jordan，F.等人（2003）J. Am. 125，12732-12738）。因此，我们认为所有四面体ThDP结合的共价复合物也将优选这种互变异构体，并且ThDP的4 '-氨基嘧啶参与ThDP酶的酸碱催化的多个步骤。YPDC和PDHc-E1及其活性中心变体的研究，结合化学模型，能够将330 nm处的负带分配给ThDP的4 '-氨基嘧啶互变异构体（假定的电子供体）和噻唑鎓环（假定的电子受体）之间的电荷转移跃迁，蛋白质的任何氨基酸侧链都没有显著贡献。然而，在YPDC和PDHc-E1中，需要底物或底物替代物的存在才能进行检测，这表明在320-330 nm处的条带可用作两种酶上涉及氨基互变异构体的米氏复合物的报告物。由于300 nm附近的正带报告了ThDP的1 '，4'-亚氨基互变异构体，因此现在可以使用动力学监测两种互变异构形式的方法。
Two circular dichroism signals observed on thiamin diphosphate (ThDP)-dependent enzymes, a positive band in the 300-305 nm range and a negative one in the 320-330 nm range, were investigated on yeast pyruvate decarboxylase (YPDC) and on the E1 subunit of the Escherichia coli pyruvate dehydrogenase complex (PDHc-E1). Addition of the tetrahedral ThDP-acetaldehyde adduct, 2-alpha-hydroxyethylThDP, to PDHc-E1 generates the positive band at 300 nm, consistent with the formation of the 1',4'-iminopyrimidine tautomer, as also demonstrated for phosphonolactylthiamin diphosphate, a stable analogue of the tetrahedral ThDP-pyruvate adduct 2-alpha-lactylThDP (Jordan, F. et al. (2003) J. Am. Chem. Soc. 125, 12732-12738). Therefore, we suggest that all tetrahedral ThDP-bound covalent complexes will also prefer this tautomer, and that the 4'-aminopyrimidine of ThDP participates in multiple steps of acid-base catalysis on ThDP enzymes. Studies with YPDC and PDHc-E1, and their active center variants, in conjunction with chemical models, enabled assignment of the negative band at 330 nm to a charge-transfer transition between the 4'-aminopyrimidine tautomer (presumed electron donor) and the thiazolium ring (presumed electron acceptor) of ThDP, with no significant contributions from any amino acid side chain of the proteins. However, in both YPDC and PDHc-E1, the presence of substrate or substrate surrogate was required to enable detection, suggesting that the band at 320-330 nm be used as a reporter for the Michaelis complex, involving the amino tautomer, on both enzymes. As the positive band near 300 nm reports on the 1',4'-imino tautomer of ThDP, methods are now available for kinetic monitoring of both tautomeric forms.