Alternate substrates of human glutaryl-CoA dehydrogenase: structure and reactivity of substrates, and identification of a novel 2-enoyl-CoA product.
Alternate substrates of human glutaryl-CoA dehydrogenase: structure and reactivity of substrates, and identification of a novel 2-enoyl-CoA product.
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人戊二酰辅酶 A 脱氢酶的替代底物：底物的结构和反应性，以及新型 2-烯酰辅酶 A 产物的鉴定。
DOI：
10.1021/bi015617n
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发表时间：
2002
期刊：
影响因子：
2.9
通讯作者：
Frerman,FrankE
中科院分区：
文献类型：
--
作者：
Rao,KSudhindra;VanderVelde,David;Dwyer,TimothyM;Goodman,StephenI;Frerman,FrankE
使用一系列替代底物研究了人戊二酰辅酶A脱氢酶催化的脱氢反应。这些底物在 γ 位具有各种取代基，取代了生理底物戊二酰辅酶 A 的羧酸盐。测定了六种替代底物的稳态动力学常数以及厌氧半反应中黄素还原的程度。这些底物之一，4-硝基丁酰辅酶A，以前被认为不是脱氢酶的底物；然而，当六氟磷酸二茂铁 (FcPF6) 为电子受体时，该酶确实会用 akcat 氧化这种底物类似物，氧化程度低于戊二酰辅酶 A 的 2%。用 4-硝基丁酰辅酶 A 对脱氢酶进行无氧滴定，表明黄素没有减少；但在 460 nm 区域显示吸光度增加，表明类似物去质子化形成 α-碳负离子。这些数据的分析表明结合化学计量约为1.0。在有氧条件下，观察到第二个吸收最大值，λmax= 366 nm。后一种发色团的生成依赖于电子受体（O2 或 FcPF6），并且催化碱 Glu370 极大地促进了这一过程。 466 nm 吸收物质仍然与酶结合，而 366 nm 吸收物质仅存在于溶液中。通过质谱、1H NMR和化学分析，后一种化合物被鉴定为4-硝基-丁-2-烯酰基-CoA。产生硝基的酶产物 4-硝基-丁-2-烯酰基-CoA 的电离发生在溶液中，而不是在酶上。 kcat 随取代基性质的变化表明，不同的取代基会影响脱氢的活化自由能 ΔG⧧。 log(kcat) 与γ-取代基的场效应参数F 之间存在良好的相关性。使用定量结构-活性关系（QSAR）未发现任何其他动力学或平衡常数与取代基参数之间的相关性。 4-硝基丁酰-CoA 是在 γ 位具有强吸电子硝基的极端例子。
The dehydrogenation reaction catalyzed by human glutaryl-CoA dehydrogenase was investigated using a series of alternate substrates. These substrates have various substituents at the γ position in place of the carboxylate of the physiological substrate, glutaryl-CoA. The steady-state kinetic constants of the six alternate substrates and the extent of flavin reduction in the anaerobic half-reaction were determined. One of these substrates, 4-nitrobutyryl-CoA, was previously thought not to be a substrate of the dehydrogenase; however, the enzyme does oxidize this substrate analogue with akcatthat is less than 2% of that with glutaryl-CoA when ferrocenium hexafluorophosphate (FcPF6) is the electron acceptor. Anaerobic titration of the dehydrogenase with 4-nitrobutyryl-CoA showed no reduction of the flavin; but instead showed an increased absorbance in the 460 nm region suggesting deprotonation of the analogue to form the α-carbanion. Analysis of these data indicated a binding stoichiometry of about 1.0. Under aerobic conditions, a second absorption maximum is observed with λmax= 366 nm. The generation of the latter chromophore is dependent on an electron acceptor, either O2or FcPF6, and is greatly facilitated by the catalytic base Glu370. The 466 nm absorbing species remains enzyme-bound while the 366 nm absorbing species is present only in solution. The latter compound was identified as 4-nitronate-but-2-enoyl-CoA by mass spectrometry,1H NMR, and chemical analyses. Ionization of the enzymatic product, 4-nitro-but-2-enoyl-CoA, that yields the nitronate occurs in solution and not on the enzyme. The variation ofkcatwith the nature of the substituent suggests that the various substituents affect the free energy of activation, ΔG⧧, for dehydrogenation. There is a good correlation between log(kcat) andF, the field effect parameter, of the γ-substituent. No correlation was found between any other kinetic or equilibrium constants and the substituent parameters using quantitative structure−activity relationships (QSAR). 4-Nitrobutyryl-CoA is the extreme example with the strongly electron-withdrawing nitro group in the γ position.