Collins J H, Reed L J
Proc Natl Acad Sci U S A. 1977 Oct;74(10):4223-7. doi: 10.1073/pnas.74.10.4223.
The dihydrolipoyl transacetylase component of the Escherichia coli pyruvate dehydrogenase complex [pyruvate:lipoate oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1] bears two sites on each of its 24 polypeptide chains that undergo reductive acetylation by [2-(14)C]pyruvate and thiamin pyrophosphate, acetylation by [1-(14)C]acetyl-CoA in the presence of DPNH, and reaction with N-ethyl[2,3-(14)C]maleimide in the presence of pyruvate and thiamin pyrophosphate. The data strongly imply that these sites are covalently bound lipoyl moieties. The results of similar experiments with the E. coli alpha-ketoglutarate dehydrogenase complex [2-oxoglutarate:lipoate oxidoreductase (decarboxylating and acceptor-succinylating), EC 1.2.4.2] indicate that its dihydrolipoyl transsuccinylase component bears only one lipoyl moiety on each of its 24 chains. Charging of the 48 acetyl acceptor sites on the transacetylase or the 24 succinyl acceptor sites on the transsuccinylase by pyruvate or alpha-ketoglutarate, respectively, and thiamin pyrophosphate was observed in the presence of only a few functionally active pyruvate dehydrogenase or alpha-ketoglutarate dehydrogenase chains. Extensive crosslinking of the transacetylase chains was observed when the pyruvate dehydrogenase complex was treated with pyruvate and thiamin pyrophosphate or with DPNH in the presence of N,N'-o- or N,N'-p-phenylenedimaleimide, respectively. When the alpha-ketoglutarate dehydrogenase complex was treated with DPNH in the presence of N,N'-p-phenylenedimaleimide, only transsuccinylase monomers and crosslinked transsuccinylase dimers were detected. It appears that the 48 lipoyl moieties in the transacetylase and the 24 lipoyl moieties in the transsuccinylase comprise an interacting network that functions as an acyl group and electron pair relay system through thiol-disulfide and acyl-transfer reactions among all of the lipoyl moieties.
大肠杆菌丙酮酸脱氢酶复合体[丙酮酸：硫辛酸氧化还原酶（脱羧并乙酰基化受体），EC 1.2.4.1]的二氢硫辛酰转乙酰基酶组分在其24条多肽链的每条链上都有两个位点，这些位点可被[2-(14)C]丙酮酸和硫胺焦磷酸进行还原乙酰化，在DPNH存在下被[1-(14)C]乙酰辅酶A乙酰化，并在丙酮酸和硫胺焦磷酸存在下与N-乙基[2,3-(14)C]马来酰亚胺反应。这些数据有力地表明这些位点是共价结合的硫辛酰部分。用大肠杆菌α-酮戊二酸脱氢酶复合体[2-氧代戊二酸：硫辛酸氧化还原酶（脱羧并琥珀酰基化受体），EC 1.2.4.2]进行类似实验的结果表明，其二氢硫辛酰转琥珀酰基酶组分在其24条链的每条链上仅带有一个硫辛酰部分。在仅存在少数具有功能活性的丙酮酸脱氢酶或α-酮戊二酸脱氢酶链的情况下，分别观察到丙酮酸或α-酮戊二酸以及硫胺焦磷酸对转乙酰基酶上的48个乙酰基受体位点或转琥珀酰基酶上的24个琥珀酰基受体位点的加载。当丙酮酸脱氢酶复合体分别用丙酮酸和硫胺焦磷酸处理或在N,N'-邻苯二甲酰亚胺或N,N'-对苯二甲酰亚胺存在下用DPNH处理时，观察到转乙酰基酶链的广泛交联。当α-酮戊二酸脱氢酶复合体在N,N'-对苯二甲酰亚胺存在下用DPNH处理时，仅检测到转琥珀酰基酶单体和交联的转琥珀酰基酶二聚体。看来，转乙酰基酶中的48个硫辛酰部分和转琥珀酰基酶中的24个硫辛酰部分构成了一个相互作用的网络，该网络通过所有硫辛酰部分之间的硫醇-二硫键和酰基转移反应作为酰基和电子对中继系统发挥作用。
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