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首页> 外文期刊>Journal of Molecular Biology >RECOGNITION OF A SURFACE LOOP OF THE LIPOYL DOMAIN UNDERLIES SUBSTRATE CHANNELLING IN THE PYRUVATE DEHYDROGENASE MULTIENZYME COMPLEX
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RECOGNITION OF A SURFACE LOOP OF THE LIPOYL DOMAIN UNDERLIES SUBSTRATE CHANNELLING IN THE PYRUVATE DEHYDROGENASE MULTIENZYME COMPLEX

机译：丙酮酸脱氢酶多酶复合物中底物通道的脂环族表面环的识别

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摘要

In the pyruvate dehydrogenase multienzyme complex of Bacillus stearother-mophilus, the interaction between the pyruvate decarboxylase (E1p) component and the lipoyl domain of the dihydrolipoyl acetyltransferase (E2) component was investigated using a combination of site-directed mutagenesis and NMR spectroscopy. Residues 11 to 15 (EGIHE) of the lipoyl domain, part of a surface loop dose in space to the beta-turn containing the lipoyl-lysine residue (position 42), were deleted or replaced. The mutant domains all retained their three-dimensional structures and ability to become lipoylated, but in the absence of the loop the lipoyl-lysine residue could no longer be reductively acetylated by E1p. A mutation (N40A) in the N-terminal part of the lipoyl-lysine hairpin showed that it is involved in recognition of the domain by E1p but other mutations in the loop (E15A) and close to the lipoyl-lysine hairpin (V44S, V45S and E46A) were without effect. The heteronuclear multiple quantum coherence NMR spectra of N-15-labelled lipoyl domain in the presence and absence of B. stearothermophilus E1p were recorded. Of the 85 amino acid residues in the lipoyl domain, 13 exhibited significant differences in chemical shift. These differences, most of which were associated with residues in the surface loop between positions 8 and 15 and in, or close to, the lipoyl-lysine hairpin, indicate that E1p makes contact with the lipoyl domain in these areas. The combined results of directed mutagenesis and NMR spectroscopy point to the surface loop as a major determinant of the interaction of lipoyl domain with E1p. The specificity of this essential interaction provides the molecular basis of substrate channelling in this, the first committed, step of the enzyme reaction mechanism. (C) 1996 Academic Press Limited [References: 40]
机译：在硬脂芽孢杆菌-嗜热芽孢杆菌的丙酮酸脱氢酶多酶复合物中，结合定点诱变和NMR光谱研究了丙酮酸脱羧酶（E1p）组分与二氢脂酰乙酰基转移酶（E2）组分的脂酰结构域之间的相互作用。删除或替换了脂酰结构域的残基11至15（EGIHE），这是与含有脂酰赖氨酸残基（位置42）的β-转角在空间上的表面环剂量的一部分。突变域都保留了其三维结构，并具有脂酰化的能力，但是在没有环的情况下，脂酰赖氨酸残基不能再被E1p还原乙酰化。脂酰赖氨酸发夹的N端部分的突变（N40A）表明，它参与了E1p对结构域的识别，但环中有其他突变（E15A）并靠近脂酰赖氨酸发夹（V44S，V45S和E46A）无效。记录在存在和不存在嗜热脂肪芽孢杆菌E1p的情况下N-15标记的脂酰结构域的异核多量子相干NMR谱。在脂酰结构域的85个氨基酸残基中，有13个在化学位移上显示出显着差异。这些差异（大部分与位置8和15之间以及在脂酰赖氨酸发夹中或附近的表面环中的残基相关）表明E1p在这些区域与脂酰结构域接触。定向诱变和NMR光谱的结合结果表明，表面环是脂酰结构域与E1p相互作用的主要决定因素。这种必不可少的相互作用的特异性为酶反应机理这一第一步中的底物通道化提供了分子基础。 （C）1996 Academic Press Limited [参考号：40]

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