The effect of a Glu370Asp mutation in glutaryl-CoA dehydrogenase on proton transfer to the dienolate intermediate.
The effect of a Glu370Asp mutation in glutaryl-CoA dehydrogenase on proton transfer to the dienolate intermediate.
复制标题
戊二酰辅酶A脱氢酶中的 Glu370Asp 突变对质子转移到二烯醇盐中间体的影响。
DOI：
10.1021/bi7009597
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发表时间：
2007
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;Fu,Zhuji;Albro,Mark;Narayanan,Beena;Baddam,Saritha;Lee,Hyun-JooK;Kim,Jung-JaP;Frerman,FrankE
我们已经确定了稳态速率常数和净速率常数的化学步骤中催化的戊二酰辅酶A脱氢酶的E370 D突变体，并将它们与野生型脱氢酶。我们寻求这些速率常数的变化的突变体的结构与替代基板，4-硝基丁酸共结晶的理由。用天冬氨酸取代催化碱E370，导致3-硫代戊二酰-CoA的C-2处质子提取速率常数降低24%，因为配体的C-2与残基370处最接近的羧基氧之间的距离从2.9 nm增加到3.1 nm。由于突变，从天然底物的C-3到黄素的N5的氢化物转移（包括C-2处的质子提取）导致黄素还原的净速率常数降低了81%，尽管距离仅增加了0.7 μ m。与3-thiaglutaryl-CoA的烯醇化物、还原半反应（还原黄素与氧化形式的底物）和脱羧后的二烯醇化物相关的电荷转移带的强度显著减小。结构研究表明，底物与异咯嗪的S-C1（O）-C2平面的距离增加和角度变化，大大改变了还原和氧化半反应的速率。活性位点几何结构的这种变化也改变了四碳二烯醇化物中间体的质子化位置，以产生动力学上有利的产物乙烯基乙酰基-CoA，其进一步异构化为药物稳定的正常产物巴豆酰-CoA。
We have determined steady-state rate constants and net rate constants for the chemical steps in the catalytic pathway catalyzed by the E370D mutant of glutaryl-CoA dehydrogenase and compared them with those of the wild-type dehydrogenase. We sought rationales for changes in these rate constants in the structure of the mutant cocrystallized with the alternate substrate, 4-nitrobutyric acid. Substitution of aspartate for E370, the catalytic base, results in a 24% decrease in the rate constant for proton abstraction at C-2 of 3-thiaglutaryl-CoA as the distance between C-2 of the ligand and the closest carboxyl oxygen at residue 370 increases from 2.9 Å to 3.1 Å. The net rate constant for flavin reduction due to hydride transfer from C-3 of the natural substrate, which includes proton abstraction at C-2, to N5 of the flavin decreases by 81% due to the mutation, although the distance increases only by 0.7 Å. The intensities of charge-transfer bands associated with the enolate of 3-thiaglutaryl-CoA, the reductive half-reaction (reduced flavin with oxidized form of substrate), and the dienolate following decarboxylation are considerably diminished. Structural investigation suggests that the increased distance and the change in angle of the S−C1(O)−C2 plane of the substrate with the isoalloxazine substantially alter rates of the reductive and oxidative half-reactions. This change in active site geometry also changes the position of protonation of the four carbon dienolate intermediate to produce kinetically favorable product, vinylacetyl-CoA, which is further isomerized to the thermodynamically stable normal product, crotonyl-CoA.