Burns B P, Hazell S L, Mendz G L
School of Microbiology, University of New South Wales, Sydney, Australia.
Microbiology (Reading). 1995 Dec;141 ( Pt 12):3113-8. doi: 10.1099/13500872-141-12-3113.
A biotinylated acetyl-CoA carboxylase from the microaerophilic bacterium Helicobacter pylori was partially purified and characterized. The approximate molecular mass of the native enzyme was estimated at 235 kDa by native PAGE. A single band corresponding to approximately 24 kDa was detected by SDS-PAGE, suggesting that the native enzyme is a multi-protein complex. The protein was isolated from the soluble fraction of the cell. Catalytic activity was acetyl-CoA-dependent and inhibited by avidin but unaffected by avidin pretreated with excess biotin. The end-product of the reaction was identified as malonyl-CoA and the reaction was shown to be reversible by NMR spectroscopy. The activity of the enzyme was 0.29 mumol min-1 (mg protein)-1. The Vmax for bicarbonate was calculated at 0.73 mumol min-1 (mg protein)-1, and the affinity of the enzyme for this substrate was relatively low, with an apparent Km of 16.6 mM. These data provide the first evidence of a possible physiological role for the requirement of high levels of CO2 for growth in vitro of this bacterium.
对来自微需氧细菌幽门螺杆菌的一种生物素化乙酰辅酶A羧化酶进行了部分纯化和表征。通过非变性聚丙烯酰胺凝胶电泳（native PAGE）估计天然酶的近似分子量为235 kDa。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）检测到一条对应于约24 kDa的单条带，表明天然酶是一种多蛋白复合物。该蛋白质从细胞的可溶性部分中分离出来。催化活性依赖于乙酰辅酶A，并且被抗生物素蛋白抑制，但不受用过量生物素预处理的抗生物素蛋白的影响。反应的终产物被鉴定为丙二酰辅酶A，并且通过核磁共振光谱显示该反应是可逆的。该酶的活性为0.29 μmol·min⁻¹·(mg蛋白质)⁻¹。碳酸氢盐的最大反应速度（Vmax）计算为0.73 μmol·min⁻¹·(mg蛋白质)⁻¹，并且该酶对该底物的亲和力相对较低，表观米氏常数（Km）为16.6 mM。这些数据首次证明了该细菌体外生长需要高水平二氧化碳可能具有的生理作用。
