首页> 外文OA文献 >Amino acid sequence analysis of the lipoyl and peripheral subunit-binding domains in the lipoate acetyltransferase component of the pyruvate dehydrogenase complex from Bacillus stearothermophilus.
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Amino acid sequence analysis of the lipoyl and peripheral subunit-binding domains in the lipoate acetyltransferase component of the pyruvate dehydrogenase complex from Bacillus stearothermophilus.

机译:嗜热脂肪芽孢杆菌丙酮酸脱氢酶复合物的脂酸酯乙酰基转移酶组分中的脂酰和外围亚基结合域的氨基酸序列分析。

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摘要

The pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus comprises a structural core, composed of 60 dihydrolipoamide acetyltransferase (E2p) subunits, which binds multiple copies of pyruvate decarboxylase (E1p) and dihydrolipoamide dehydrogenase (E3) subunits. After limited proteolysis with chymotrypsin, the N-terminal lipoyl domain of E2p was excised, purified and sequenced. The residual complex, which remained assembled, was then digested with trypsin under mild conditions. This treatment promoted complete disassembly of the complex and the various components were separated by gel filtration and h.p.l.c. A folded fragment of E2p containing about 50 amino acid residues was identified as being responsible for binding the E3 subunits, although, unlike the corresponding region of the E2p or E2o chains of the pyruvate dehydrogenase or 2-oxoglutarate dehydrogenase complexes from Escherichia coli, the fragment also bound E1p molecules. Further peptide purification and sequence analysis allowed the determination of the first 211 amino acid residues of the B. stearothermophilus E2p chain, thus providing the complete primary structure of the lipoyl domain, the E1p/E3-binding domain and the regions of polypeptide chain, probably highly flexible in nature, that link the domains to each other and to the inner-core (E2p-binding) domain. Several of the proteolytically sensitive sites were also identified. The sequence of the B. stearothermophilus E2p chain shows close homology with the sequences of the E2p and E2o chains from E. coli, although significant differences in structure are apparent. Detailed evidence for the sequence of the peptides obtained by limited proteolysis and further chemical and enzymic cleavages have been deposited as Supplementary Publication SUP 50142 (11 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 6BQ, U.K., from whom copies may be obtained as indicated in Biochem. J. (1988) 249, 5.
机译:嗜热脂肪芽孢杆菌的丙酮酸脱氢酶多酶复合物包含一个结构核心,该结构核心由60个二氢脂酰胺乙酰基转移酶(E2p)亚基组成,该亚基结合了多个拷贝的丙酮酸脱羧酶(E1p)和二氢脂酰胺脱氢酶(E3)亚基。用胰凝乳蛋白酶进行有限的蛋白水解后,切除,纯化和测序E2p的N末端脂酰结构域。然后在温和的条件下用胰蛋白酶消化仍保持组装状态的残留复合物。这种处理促进了复合物的完全分解,并且通过凝胶过滤和h.p.l.c分离了各种组分。尽管与大肠杆菌的丙酮酸脱氢酶或2-氧戊二酸脱氢酶复合物的E2p或E2o链的相应区域不同,该片段被鉴定为包含约50个氨基酸残基的E2p折叠片段与该E3亚基结合。还结合了E1p分子。进一步的肽纯化和序列分析使得可以确定嗜热脂肪芽孢杆菌E2p链的前211个氨基酸残基,从而提供了脂酰结构域,E1p / E3结合结构域和多肽链区域的完整一级结构,可能本质上是高度灵活的,它们将域彼此链接到内核(E2p绑定)域。还确定了几个蛋白水解敏感位点。尽管嗜热脂肪芽孢杆菌E2p链的序列与大肠杆菌的E2p和E2o链的序列显示出紧密的同源性,但结构上的明显差异却很明显。通过有限的蛋白水解以及进一步的化学和酶促切割获得的肽序列的详细证据已作为补充出版物SUP 50142(11页)保存在英国西约克郡LS23 6BQ韦瑟比,波士顿温泉大学,大英图书馆借阅处。可以按照《生物化学》中的说明获得副本。 J.(1988)249,5。

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