Vancura A, Vancurová I, Volc J, Jones S K, Flieger M, Basarová G, Bĕhal V
Prague Institute of Chemical Technology, Czechoslovak Academy of Sciences.
Eur J Biochem. 1989 Jan 15;179(1):221-7. doi: 10.1111/j.1432-1033.1989.tb14544.x.
Alanine dehydrogenase was purified to homogeneity from a cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1180-fold to give a 21% yield, using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The relative molecular mass of the native enzyme was determined to be 210,000 or 205,000 by equilibrium ultracentrifugation or gel filtration, respectively. The enzyme is composed of four subunits, each of Mr 51,000. Using analytical isoelectric focusing the isoelectric point of alanine dehydrogenase was found to be 6.1. The Km were 10.0 mM for L-alanine and 0.18 mM for NAD+. Km values for reductive amination were 0.23 mM for pyruvate, 11.6 mM for NH4+ and 0.05 mM for NADH. Oxidative deamination of L-alanine proceeds through a sequential-ordered binary-ternary mechanism in which NAD+ binds first to the enzyme, followed by alanine, and products are released in the order ammonia, pyruvate and NADH.
从产生泰乐菌素的弗氏链霉菌无细胞提取物中纯化得到了均一的丙氨酸脱氢酶。使用疏水色谱和离子交换快速蛋白质液相色谱相结合的方法，该酶被纯化了1180倍，产率为21%。通过平衡超速离心法和凝胶过滤法分别测定天然酶的相对分子质量为210,000或205,000。该酶由四个亚基组成，每个亚基的相对分子质量为51,000。使用分析等电聚焦法发现丙氨酸脱氢酶的等电点为6.1。L-丙氨酸的Km值为10.0 mM，NAD⁺的Km值为0.18 mM。还原胺化反应中丙酮酸的Km值为0.23 mM，NH₄⁺的Km值为11.6 mM，NADH的Km值为0.05 mM。L-丙氨酸的氧化脱氨反应通过顺序有序的二元-三元机制进行，其中NAD⁺首先与酶结合，然后是丙氨酸，产物按氨、丙酮酸和NADH的顺序释放。
