Catalysis of transthiolacylation in the active centers of dihydrolipoamide acyltransacetylase components of 2-oxo acid dehydrogenase complexes.
Catalysis of transthiolacylation in the active centers of dihydrolipoamide acyltransacetylase components of 2-oxo acid dehydrogenase complexes.
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DOI：
10.1002/2211-5463.12431
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发表时间：
2018-06
期刊：
影响因子：
2.6
通讯作者：
Jordan F
中科院分区：
文献类型：
--
作者：
Chakraborty J;Nemeria NS;Farinas E;Jordan F
大肠杆菌 2-酮戊二酸脱氢酶复合物 (OGDHc) 包含三种酶（E1o、E2o 和 E3）的多个拷贝，并且转硫酯化作用发生在 E2o 的催化域内。 S8-琥珀酰二氢硫辛酰-E2o 硫醇酯中的琥珀酰基转移至辅酶 A (CoA) 的硫醇基，形成最重要的琥珀酰-CoA。在这里，我们报告了 OGDHc 酶促转硫酯化的机制研究。证据证明 E2o 中 His375 和 Asp374 对于琥珀酰转移反应的重要性。这些残基提供的速率加速幅度（丙氨酸取代时各残基的 54 倍）表明通过形成两个氢键在对称四面体含氧阴离子中间体的稳定中发挥作用，而不是在酸碱催化中发挥作用。 His375 的位点饱和诱变研究（His375Trp 取代几乎没有惩罚）和 Asp374 上其他潜在氢键参与者的取代提供了进一步的证据，证明了其在酸碱催化中的作用。考虑到 E1o 和 2-氧戊二酸 (99 s−1) 对 E2o 硫辛酰结构域 (LDo) 进行还原性琥珀酰化的速率常数大约是整个反应（NADH 产生）中 k cat 48 s−1 的速率常数的两倍，可以得出结论，琥珀酰转移到 CoA 并释放琥珀酰-CoA，而不是还原性琥珀酰化。限速步骤。结果表明，2-含氧酸脱氢酶复合物超家族中酰基转移的催化机制得到了修正，从而提供了有关酰基辅酶A形成的基本信息，这对于包括蛋白质赖氨酸翻译后琥珀酰化在内的多种生物过程非常重要。 2-氧化戊二酸脱氢酶（http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/2/4/2.html）；二氢硫辛酰胺琥珀酰转移酶(http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/3/1/61.html)；二氢硫辛酰胺脱氢酶(http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/8/1/4.html)；丙酮酸脱氢酶(http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/2/4/1.html)；二氢硫辛酰胺乙酰转移酶（http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/3/1/12.html）。
The Escherichia coli 2‐oxoglutarate dehydrogenase complex (OGDHc) comprises multiple copies of three enzymes—E1o, E2o, and E3—and transthioesterification takes place within the catalytic domain of E2o. The succinyl group from the thiol ester of S8‐succinyldihydrolipoyl‐E2o is transferred to the thiol group of coenzyme A (CoA), forming the all‐important succinyl‐CoA. Here, we report mechanistic studies of enzymatic transthioesterification on OGDHc. Evidence is provided for the importance of His375 and Asp374 in E2o for the succinyl transfer reaction. The magnitude of the rate acceleration provided by these residues (54‐fold from each with alanine substitution) suggests a role in stabilization of the symmetrical tetrahedral oxyanionic intermediate by formation of two hydrogen bonds, rather than in acid–base catalysis. Further evidence ruling out a role in acid–base catalysis is provided by site‐saturation mutagenesis studies at His375 (His375Trp substitution with little penalty) and substitutions to other potential hydrogen bond participants at Asp374. Taking into account that the rate constant for reductive succinylation of the E2o lipoyl domain (LDo) by E1o and 2‐oxoglutarate (99 s−1) was approximately twofold larger than the rate constant for k cat of 48 s−1 for the overall reaction (NADH production), it could be concluded that succinyl transfer to CoA and release of succinyl‐CoA, rather than reductive succinylation, is the rate‐limiting step. The results suggest a revised mechanism of catalysis for acyl transfer in the superfamily of 2‐oxo acid dehydrogenase complexes, thus provide fundamental information regarding acyl‐CoA formation, so important for several biological processes including post‐translational succinylation of protein lysines. 2‐oxoglutarate dehydrogenase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/2/4/2.html); dihydrolipoamide succinyltransferase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/3/1/61.html); dihydrolipoamide dehydrogenase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/8/1/4.html); pyruvate dehydrogenase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/2/4/1.html); dihydrolipoamide acetyltransferase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/3/1/12.html).
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影响因子：
7.4
作者：
Jung, Sung Yun;Li, Yehua;Qin, Jun
通讯作者：
Qin, Jun
影响因子：
11.4
作者：
Kato, Masato;Wynn, R. Max;Chuang, David T.
通讯作者：
Chuang, David T.
影响因子：
5.6
作者：
Knapp, JE;Mitchell, DT;Hackert, ML
通讯作者：
Hackert, ML
DOI：
10.1073/pnas.83.11.3743
发表时间：
1986-06-01
影响因子：
11.1
作者：
BRYAN, P;PANTOLIANO, MW;POULOS, T
通讯作者：
POULOS, T
影响因子：
5.6
作者：
MATTEVI, A;OBMOLOVA, G;HOL, WGJ
通讯作者：
HOL, WGJ