Arfman N, Van Beeumen J, De Vries G E, Harder W, Dijkhuizen L
Department of Microbiology, University of Groningen, Haren, The Netherlands.
J Biol Chem. 1991 Feb 25;266(6):3955-60.
All thermotolerant methanol-utilizing Bacillus spp. investigated by us possess a NAD-dependent methanol dehydrogenase (MDH) activity which is stimulated by a protein present in the soluble fraction of Bacillus sp. C1 cells. This activator protein was purified to homogeneity from Bacillus sp. C1 cells grown at a low dilution rate in a methanol-limited chemostat culture. The native activator protein (Mr = 50,000) is a dimer of Mr = 27,000 subunits. The N-terminal amino acid sequence revealed no significant similarity with any published sequences. Stimulation of MDH activity by the activator protein required the presence of Mg2+ ions. Plots of specific MDH activity versus activator protein concentration revealed Michaelis-Menten type kinetics. In the presence of activator protein, MDH displayed biphasic kinetics (v versus substrate concentration) toward C1-C4 primary alcohols and NAD. The data suggest that in the presence of activator protein plus Mg2+ ions, MDH possesses a high affinity active site for alcohols and NAD, in addition to an activator- and Mg2(+)-independent low affinity active site. The activation mechanism remains to be elucidated.
我们研究的所有耐热性利用甲醇的芽孢杆菌属细菌都具有一种依赖NAD的甲醇脱氢酶（MDH）活性，该活性受到芽孢杆菌属C1菌株细胞可溶部分中一种蛋白质的刺激。这种激活蛋白是从在甲醇限制恒化器培养中以低稀释率生长的芽孢杆菌属C1菌株细胞中纯化至同质的。天然激活蛋白（Mr = 50,000）是由Mr = 27,000亚基组成的二聚体。其N端氨基酸序列与任何已发表序列均无明显相似性。激活蛋白对MDH活性的刺激需要Mg2+离子的存在。特定MDH活性与激活蛋白浓度的关系图显示出米氏动力学。在存在激活蛋白的情况下，MDH对C1 - C4伯醇和NAD呈现双相动力学（v与底物浓度）。数据表明，在存在激活蛋白和Mg2+离子的情况下，除了一个不依赖激活剂和Mg2+的低亲和力活性位点外，MDH还具有一个对醇和NAD的高亲和力活性位点。激活机制仍有待阐明。
