Interaction of 3,4-dienoyl-CoA thioesters with medium chain acyl-CoA dehydrogenase: stereochemistry of inactivation of a flavoenzyme.
Interaction of 3,4-dienoyl-CoA thioesters with medium chain acyl-CoA dehydrogenase: stereochemistry of inactivation of a flavoenzyme.
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3,4-二烯酰基辅酶A硫酯与中链酰基辅酶A脱氢酶的相互作用：黄素酶失活的立体化学。
DOI：
10.1021/bi0109818
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发表时间：
2001
期刊：
影响因子：
2.9
通讯作者：
Thorpe,C
中科院分区：
文献类型：
--
作者：
Wang,W;Fu,Z;Zhou,JZ;Kim,JJ;Thorpe,C
中链酰基辅酶 A 脱氢酶可被外消旋 3,4-二烯酰基辅酶 A 衍生物快速抑制，每种酶黄素的化学计量为两分子外消旋体。 R-和S-3，4-癸二烯酰辅酶A的合成表明R-对映异构体是该酶的有效的化学计量抑制剂。在加合物形成之前，α-质子提取产生烯醇化物至氧化黄素电荷转移中间体。还原的、无活性的酶的晶体结构显示出连接 2,4-二烯酰基-CoA 部分的 C-4 碳和还原型黄素的 N5 位点的单个共价键。通过释放共轭 2,4-二烯来逆转加合物形成的动力学被评估为酰基链长度和 CoA 部分截短的函数。该加合物与中链长度联烯抑制剂最稳定。然而，R-3,4-癸二烯酰基-泛硫氨酸和-N-乙酰半胱胺的加合物的动力学稳定性比全长 CoA 硫酯高约 9 倍和 >100 倍。这些还原酶种类的晶体结构确定为 2.4 Å，表明氢键与抑制剂羰基氧的位置以及催化碱基的位置是加合物稳定性的重要决定因素。 S-3,4-癸二烯酰辅酶A不是中链脱氢酶的显着抑制剂并且不形成可检测的黄素加合物。然而，S-异构体迅速异构化为反-反-2，4-共轭二烯。蛋白质模型研究表明，S-对映体不能足够接近异咯嗪环以形成黄素加合物，但可以通过催化碱轻松地再质子化。这些研究表明，辅酶A硫酯的截短可能允许设计出意想不到的强效亲脂性脂肪酸氧化抑制剂。
The medium chain acyl-CoA dehydrogenase is rapidly inhibited by racemic 3,4-dienoyl-CoA derivatives with a stoichiometry of two molecules of racemate per enzyme flavin. Synthesis ofR- andS-3,4-decadienoyl-CoA shows that theR-enantiomer is a potent, stoichiometric, inhibitor of the enzyme. α-Proton abstraction yields an enolate to oxidized flavin charge-transfer intermediate prior to adduct formation. The crystal structure of the reduced, inactive enzyme shows a single covalent bond linking the C-4 carbon of the 2,4-dienoyl-CoA moiety and the N5 locus of reduced flavin. The kinetics of reversal of adduct formation by release of the conjugated 2,4-diene were evaluated as a function of both acyl chain length and truncation of the CoA moiety. The adduct is most stable with medium chain length allenic inhibitors. However, the adducts withR-3,4-decadienoyl-pantetheine and -N-acetylcysteamine are some 9- and >100-fold more kinetically stable than the full-length CoA thioester. Crystal structures of these reduced enzyme species, determined to 2.4 Å, suggest that the placement of H-bonds to the inhibitor carbonyl oxygen and the positioning of the catalytic base are important determinants of adduct stability. TheS-3,4-decadienoyl-CoA is not a significant inhibitor of the medium chain dehydrogenase and does not form a detectable flavin adduct. However, theS-isomer is rapidly isomerized to the trans-trans-2,4-conjugated diene. Protein modeling studies suggest that theS-enantiomer cannot approach close enough to the isoalloxazine ring to form a flavin adduct, but can be facilely reprotonated by the catalytic base. These studies show that truncation of CoA thioesters may allow the design of unexpectedly potent lipophilic inhibitors of fatty acid oxidation.