Concerted and stepwise dehydration mechanisms observed in wild-type and mutated Escherichia coli dTDP-glucose 4,6-dehydratase
Concerted and stepwise dehydration mechanisms observed in wild-type and mutated Escherichia coli dTDP-glucose 4,6-dehydratase
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DOI：
10.1021/bi011748c
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发表时间：
2002-02-26
期刊：
影响因子：
2.9
通讯作者：
Frey, PA
中科院分区：
文献类型：
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作者：
Hegeman, AD;Gross, JW;Frey, PA
大肠杆菌dtdp -葡萄糖4,6-脱水酶（4,6-脱水酶）在活性位点将dtdp -葡萄糖转化为dtdp -4-酮-6-脱氧葡萄糖，分为三个步骤：脱氢成dtdp -4-酮葡萄糖，脱水成dtdp -4-酮葡萄糖-5,6-烯，C6还原为甲基。4,6-脱水酶利用紧密结合的NAD（+）作为辅酶短暂氧化底物，激活底物进行脱水步骤。脱水可能通过两种机制中的任何一种发生，dtdp -4-酮葡萄糖中间体烯醇化，然后是消除[由Gerlt， J. A.和Gassman， P. G. (1992) J. Aria提出的β消除。化学。[Soc. 114, 5928-5934]，或协同5,6-从中间体中去除水。为了确定这两种机制中的一种，采用基质辅助激光解吸/电离飞行时间质谱法同时对葡萄糖基C5（H-1/H-2）溶剂氢和C6（OH)-O-16/(OH)-O-18）溶剂氧交换进行了动力学表征。野生型酶的反应是通过协同脱水机制进行的。有趣的是，酸性催化剂Asp135对Asn或Ala的突变改变了机制，使烯醇化在不同程度上发生。虽然天冬氨酸135是野生型酶脱水的酸性催化剂，但D135N和D135A脱水酶的烯醇化能力差异表明该残基具有额外的作用。我们假设，在天冬氨酸变体中观察到的从协调脱水机制到逐步脱水机制的转换是由于对活性位点葡萄糖基C5-C6键旋转的控制丧失。
The conversion of dTDP-glucose into dTDP-4-keto-6-deoxyglucose by Escherichia coli dTDP-glucose 4,6-dehydratase (4,6-dehydratase) takes place in the active site in three steps: dehydrogenation to dTDP-4-ketoglucose, dehydration to dTDP-4-ketoglucose-5,6-ene, and rereduction of C6 to the methyl group. The 4,6-dehydratase makes use of tightly bound NAD(+) as the coenzyme for transiently oxidizing the substrate, activating it for the dehydration step. Dehydration may occur by either of two mechanisms, enolization of the dTDP-4-ketoglucose intermediate, followed by elimination [as proposed for beta-eliminations by Gerlt, J. A., and Gassman, P. G. (1992) J. Aria. Chem. Soc. 114, 5928-5934], or a concerted 5,6-elimination of water from the intermediate. To assign one of these two mechanisms, a simultaneous kinetic characterization of glucosyl C5(H-1/H-2) solvent hydrogen and C6((OH)-O-16/(OH)-O-18) solvent oxygen exchange was performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The reaction of the wild-type enzyme is shown to proceed through a concerted dehydration mechanism. Interestingly, mutation of Asp135, the acid catalyst, to Asn or Ala alters the mechanism, allowing enolization to occur to varying extents. While aspartic acid 135 is the acid catalyst for dehydration in the wild-type enzyme, the differential enolization capabilities of D135N and D135A dehydratases suggest an additional role for this residue. We postulate that the switch from a concerted to stepwise dehydration mechanism observed in the aspartic acid variants is due to the loss of control over the glucosyl C5-C6 bond rotation in the active site.