Packman L C, Perham R N, Roberts G C
Biochem J. 1984 Jan 1;217(1):219-27. doi: 10.1042/bj2170219.
The pyruvate dehydrogenase complex of Bacillus stearothermophilus was treated with Staphylococcus aureus V8 proteinase, causing cleavage of the dihydrolipoamide acetyltransferase polypeptide chain (apparent Mr 57 000), inhibition of the enzymic activity and disassembly of the complex. Fragments of the dihydrolipoamide acetyltransferase chains with apparent Mr 28 000, which contained the acetyltransferase activity, remained assembled as a particle ascribed the role of an inner core of the complex. The lipoic acid residue of each dihydrolipoamide acetyltransferase chain was found as part of a small but stable domain that, unlike free lipoamide, was able still to function as a substrate for reductive acetylation by pyruvate in the presence of intact enzyme complex or isolated pyruvate dehydrogenase (lipoamide) component. The lipoyl domain was acidic and had an apparent Mr of 6500 (by sedimentation equilibrium), 7800 (by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) and 10 000 and 20 400 (by gel filtration in the presence and in the absence respectively of 6M-guanidinium chloride). 1H-n.m.r. spectroscopy of the dihydrolipoamide acetyltransferase inner core demonstrated that it did not contain the segments of highly mobile polypeptide chain found in the pyruvate dehydrogenase complex. 1H-n.m.r. spectroscopy of the lipoyl domain demonstrated that it had a stable and defined tertiary structure. From these and other experiments, a model of the dihydrolipoamide acetyltransferase chain is proposed in which the small, folded, lipoyl domain comprises the N-terminal region, and the large, folded, core-forming domain that contains the acetyltransferase active site comprises the C-terminal region. These two regions are separated by a third segment of the chain, which includes a substantial region of polypeptide chain that enjoys high conformational mobility and facilitates movement of the lipoyl domain between the various active sites in the enzyme complex.
嗜热脂肪芽孢杆菌的丙酮酸脱氢酶复合体用金黄色葡萄球菌V8蛋白酶处理后，二氢硫辛酰胺乙酰转移酶多肽链（表观分子量57000）发生裂解，酶活性受到抑制，复合体发生解离。具有乙酰转移酶活性、表观分子量为28000的二氢硫辛酰胺乙酰转移酶链片段仍组装成一个颗粒，该颗粒被认为是复合体的内核。发现每条二氢硫辛酰胺乙酰转移酶链的硫辛酸残基是一个小而稳定结构域的一部分，与游离硫辛酰胺不同，在完整的酶复合体或分离的丙酮酸脱氢酶（硫辛酰胺）组分存在的情况下，它仍能作为丙酮酸进行还原乙酰化反应的底物。硫辛酰结构域呈酸性，其表观分子量通过沉降平衡法测定为6500，通过十二烷基硫酸钠/聚丙烯酰胺凝胶电泳法测定为7800，通过在有和没有6M盐酸胍存在下的凝胶过滤法分别测定为10000和20400。二氢硫辛酰胺乙酰转移酶内核的1H核磁共振光谱表明，它不包含丙酮酸脱氢酶复合体中发现的高度可移动多肽链片段。硫辛酰结构域的1H核磁共振光谱表明，它具有稳定且明确的三级结构。基于这些及其他实验，提出了二氢硫辛酰胺乙酰转移酶链的模型，其中小的、折叠的硫辛酰结构域包含N端区域，而包含乙酰转移酶活性位点的大的、折叠的、形成核心的结构域包含C端区域。这两个区域被链的第三段隔开，该段包括相当长的一段多肽链，其具有高构象流动性，有助于硫辛酰结构域在酶复合体的各个活性位点之间移动。
