Danson M J, Fersht A R, Perham R N
Proc Natl Acad Sci U S A. 1978 Nov;75(11):5386-90. doi: 10.1073/pnas.75.11.5386.
In the absence of CoA and presence of pyruvate, the lipoic acid residues covalently bound to the lipoate acetyltransferase core component (acetyl-CoA:dihydrolipoate S-acetyltransferase, EC 2.3.1.12) of the pyruvate dehydrogenase multienzyme complex of Escherichia coli become reductively acetylated. A study of a series of reassembled complexes varying only in their content of pyruvate decarboxylase [pyruvate:lipoate-oxidoreductase (decarboxylating and acceptor-acetylating) EC 1.2.4.1] showed that the initial direct reductive acetylation of lipoic acid residues can be followed by extensive intramolecular transacetylation reaction between lipoic acid residues on neighboring polypeptide chains of the lipoate acetyltransferase core [Bates, D. L., Danson, M. J., Hale, G., Hooper, E. A. & Perham, R. N. (1977) Nature (London) 268, 313-316]. Pulsed-quenched-flow measurements of the rates of the acetylation reactions in the various complexes now demonstrate that the intramolecular transacetylation reactions are not rate-determining in the normal reaction mechanism of the enzyme. There is therefore the potential for rapid multiple coupling of active sites in the lipoate acetyltransferase core. The rate constant for the overall complex reaction, measured by stopped-flow fluorimetry, is found to be approximately twice that for the reductive acetylation reaction measured by pulsed-quenched flow. This result could mean that CoA is an allosteric stimulator of the reductive acetylation part of the overall reaction or that there are two active sites on each chain of the lipoate acetyltransferase component working in parallel. A system of rapid functional connection of active sites in a multienzyme complex ensures that sequential reactions can be successfully coupled even under conditions of low substrate concentrations for the different steps. The substantial rate enhancement thus achieved offers a plausible explanation for the unusual complexity of the quaternary structure of the enzyme.
在没有辅酶A（CoA）而有丙酮酸存在的情况下，与大肠杆菌丙酮酸脱氢酶多酶复合体的硫辛酸乙酰转移酶核心组分（乙酰辅酶A：二氢硫辛酸S - 乙酰转移酶，EC 2.3.1.12）共价结合的硫辛酸残基会发生还原乙酰化。对一系列仅丙酮酸脱羧酶[丙酮酸：硫辛酸氧化还原酶（脱羧和受体乙酰化）EC 1.2.4.1]含量不同的重组复合体进行的研究表明，硫辛酸残基最初的直接还原乙酰化之后，相邻多肽链上硫辛酸残基之间会发生广泛的分子内转乙酰化反应，这些多肽链来自硫辛酸乙酰转移酶核心[贝茨，D.L.，丹森，M.J.，黑尔，G.，胡珀，E.A.和佩勒姆，R.N.（1977年）《自然》（伦敦）268卷，313 - 316页]。现在通过脉冲淬灭流动法对各种复合体中乙酰化反应速率的测量表明，分子内转乙酰化反应在该酶的正常反应机制中不是速率决定步骤。因此，硫辛酸乙酰转移酶核心中存在活性位点快速多重偶联的可能性。通过停流荧光法测量的整个复合体反应的速率常数，大约是通过脉冲淬灭流动法测量的还原乙酰化反应速率常数的两倍。这一结果可能意味着辅酶A是整个反应中还原乙酰化部分的变构激活剂，或者在硫辛酸乙酰转移酶组分的每条链上有两个并行工作的活性位点。多酶复合体中活性位点的快速功能连接系统确保即使在不同步骤底物浓度较低的条件下，顺序反应也能成功偶联。由此实现的显著速率增强为该酶四级结构异常复杂提供了一个合理的解释。
