Misono H, Yonezawa J, Nagata S, Nagasaki S
Department of Agricultural Chemistry, Kochi University, Japan.
J Bacteriol. 1989 Jan;171(1):30-6. doi: 10.1128/jb.171.1.30-36.1989.
NAD+-dependent phenylalanine dehydrogenase (EC 1.4.1.) was purified to homogeneity from a crude extract of Rhodococcus maris K-18 isolated from soil. The enzyme had a molecular mass of about 70,000 daltons and consisted of two identical subunits. The enzyme catalyzed the oxidative deamination of L-phenylalanine and several other L-amino acids and the reductive amination of phenylpyruvate and p-hydroxyphenylpyruvate. The enzyme required NAD+ as a natural coenzyme. The NAD+ analog 3-acetylpyridine-NAD+ showed much greater coenzyme activity than did NAD+. D-Phenylalanine, D-tyrosine, and phenylethylamine inhibited the oxidative deamination of L-phenylalanine. The enzyme reaction was inhibited by p-chloromercuribenzoate and HgCl2. Initial-velocity and product inhibition studies showed that the reductive amination proceeded through a sequential ordered ternary-binary mechanism. NADH bound first to the enzyme, followed by phenylpyruvate and then ammonia, and the products were released in the order L-phenylalanine and NAD+. The Michaelis constants were as follows: L-phenylalanine, 3.8 mM; NAD+, 0.25 mM; NADH, 43 microM; phenylpyruvate, 0.50 mM; and ammonia, 70 mM.
从土壤中分离得到的海红球菌K-18粗提物中纯化出了NAD⁺依赖性苯丙氨酸脱氢酶（EC 1.4.1.），使其达到了均一性。该酶的分子量约为70,000道尔顿，由两个相同的亚基组成。该酶催化L-苯丙氨酸和其他几种L-氨基酸的氧化脱氨反应以及苯丙酮酸和对羟基苯丙酮酸的还原胺化反应。该酶需要NAD⁺作为天然辅酶。NAD⁺类似物3-乙酰吡啶-NAD⁺的辅酶活性比NAD⁺高得多。D-苯丙氨酸、D-酪氨酸和苯乙胺抑制L-苯丙氨酸的氧化脱氨反应。该酶反应受到对氯汞苯甲酸和HgCl₂的抑制。初始速度和产物抑制研究表明，还原胺化反应通过有序的三元-二元顺序机制进行。NADH首先与酶结合，然后是苯丙酮酸，接着是氨，产物按L-苯丙氨酸和NAD⁺的顺序释放。米氏常数如下：L-苯丙氨酸为3.8 mM；NAD⁺为0.25 mM；NADH为43 μM；苯丙酮酸为0.50 mM；氨为70 mM。
