Wallis N G, Allen M D, Broadhurst R W, Lessard I A, Perham R N
Cambridge Centre for Molecular Recognition, Department of Biochemistry, University of Cambridge, UK.
J Mol Biol. 1996 Nov 1;263(3):463-74. doi: 10.1006/jmbi.1996.0589.
In the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus, 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 close 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 15N-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.
在嗜热脂肪芽孢杆菌的丙酮酸脱氢酶多酶复合物中，结合定点诱变和核磁共振光谱技术，对丙酮酸脱羧酶（E1p）组分与二氢硫辛酰胺乙酰转移酶（E2）组分的硫辛酰胺结构域之间的相互作用进行了研究。硫辛酰胺结构域中11至15位残基（EGIHE），即靠近含硫辛酰胺赖氨酸残基（42位）的β-转角的表面环的一部分，被删除或替换。突变结构域均保留了其三维结构和硫辛酰化能力，但在没有该环的情况下，硫辛酰胺赖氨酸残基不再能被E1p还原乙酰化。硫辛酰胺赖氨酸发夹N端部分的一个突变（N40A）表明它参与E1p对该结构域的识别，但环中的其他突变（E15A）以及靠近硫辛酰胺赖氨酸发夹的突变（V44S、V45S和E46A）没有影响。记录了在有和没有嗜热脂肪芽孢杆菌E1p存在的情况下，15N标记的硫辛酰胺结构域的异核多量子相干核磁共振光谱。在硫辛酰胺结构域的85个氨基酸残基中，有13个的化学位移表现出显著差异。这些差异大多与8至15位之间的表面环以及硫辛酰胺赖氨酸发夹内或其附近的残基有关，表明E1p在这些区域与硫辛酰胺结构域接触。定点诱变和核磁共振光谱的综合结果表明，表面环是硫辛酰胺结构域与E1p相互作用的主要决定因素。这种关键相互作用的特异性为该酶反应机制的第一步（即首个限速步骤）中的底物通道化提供了分子基础。
