Ohshima T, Sakane M, Yamazaki T, Soda K
Department of Chemistry, Kyoto University of Education, Japan.
Eur J Biochem. 1990 Aug 17;191(3):715-20. doi: 10.1111/j.1432-1033.1990.tb19180.x.
Alanine dehydrogenase (L-alanine: NAD+ oxidoreductase, deaminating) was simply purified to homogeneity from a thermophile, Bacillus sphaericus DSM 462, by ammonium sulfate fractionation, red-Sepharose 4B chromatography and preparative slab gel electrophoresis. The enzyme had a molecular mass of about 230 kDa and consisted of six subunits with an identical molecular mass of 38 kDa. The enzyme was much more thermostable than that from a mesophile, B. sphaericus, and retained its full activity upon heating at 75 degrees C for at least 60 min and with incubation in pH 5.5-9.5 at 75 degrees C for 10 min. The enzyme can be stored without loss of its activity in a frozen state (-20 degrees C, at pH 7.2) for over 5 months. The optimum pH for the L-alanine deamination and pyruvate amination were around 10.5 and 8.2, respectively. The enzyme exclusively catalyzed the oxidative deamination of L-alanine in the presence of NAD+, but showed low amino acceptor specificity; hydroxypyruvate, oxaloacetate, 2-oxobutyrate and 3-fluoropyruvate are also aminated as well as pyruvate in the presence of NADH and ammonia. Initial velocity and product inhibition studies showed that the reductive amination proceeded through a sequential mechanism containing partially random binding. NADH binds first to the enzyme, and then pyruvate and ammonia bind in a random fashion. The products are sequentially released from the enzyme in the order L-alanine then NAD+. A dead-end inhibition by the formation of an abortive ternary complex which consists of the enzyme, NAD+ and pyruvate was included in the reaction. A possible role of the dead-end inhibition is to prevent the enzyme from functioning in the L-alanine synthesis. The Michaelis constants for the substrates were as follows: NADH, 0.10 mM; pyruvate, 0.50 mM; ammonia, 38.0 mM; L-alanine, 10.5 mM and NAD+, 0.26 mM.
通过硫酸铵分级沉淀、红琼脂糖4B柱层析和制备性平板凝胶电泳，从嗜热菌球形芽孢杆菌DSM 462中简单纯化得到了丙氨酸脱氢酶（L-丙氨酸：NAD⁺氧化还原酶，脱氨基），使其达到了均一状态。该酶的分子量约为230 kDa，由六个分子量均为38 kDa的亚基组成。与嗜温菌球形芽孢杆菌的丙氨酸脱氢酶相比，该酶具有更高的热稳定性，在75℃加热至少60分钟以及在75℃、pH 5.5 - 9.5条件下孵育10分钟后仍能保持其全部活性。该酶在冷冻状态（-20℃，pH 7.2）下可保存5个月以上而不失活。L-丙氨酸脱氨基和丙酮酸氨基化的最适pH分别约为10.5和8.2。该酶在NAD⁺存在时专一催化L-丙氨酸的氧化脱氨基反应，但氨基受体特异性较低；在NADH和氨存在时，羟基丙酮酸、草酰乙酸、2-氧代丁酸和3-氟丙酮酸与丙酮酸一样也能被氨基化。初始速度和产物抑制研究表明，还原氨基化反应通过一种包含部分随机结合的顺序机制进行。NADH首先与酶结合，然后丙酮酸和氨以随机方式结合。产物按L-丙氨酸然后NAD⁺的顺序依次从酶上释放。反应中存在由酶、NAD⁺和丙酮酸形成的无效三元复合物导致的终产物抑制。终产物抑制的一个可能作用是防止该酶参与L-丙氨酸的合成。底物的米氏常数如下：NADH为0.10 mM；丙酮酸为0.50 mM；氨为38.0 mM；L-丙氨酸为10.5 mM；NAD⁺为0.26 mM。
