Studies on the Novel Enzyme Phenylalanine Dehydrogenase.

我们首次发现芽孢杆菌和孢子孢杆菌产生苯丙氨酸脱氢酶。我们开始与美国纽约康奈尔大学医学院的Arthur Copper教授合作。在这两年的合作中，我们取得了以下成果。(1)明确了沙氏芽孢杆菌（Bacillus shaericus）、巴氏芽孢杆菌（b.b hadius）和脲孢杆菌（Sporosarcina ureae）苯丙氨酸脱氢酶的酶学性质。利用该酶与甲酸假丝酵母脱氢酶结合，以前手性α -酮酸为原料合成了多种光学纯l -氨基酸。该酶对多种α -酮酸的还原胺化反应有效。我们已经成功地合成了天然和非天然的l -氨基酸，包括l -同苯丙氨酸。(2)我们成功合成了^<13> n标记的l -苯丙氨酸、l -酪氨酸和L-DOPA。我们已经克隆了这些酶的基因，并对其中一些基因进行了测序。它们与亮氨酸脱氢酶、链霉菌苯丙氨酸脱氢酶和谷氨酸脱氢酶具有相似性。(4)在酶筛选PKU的微孔板法中，l -苯丙氨酸被酶氧化生成苯丙酮酸，将NAD^+还原为NADH。在NADH和电子载体1-甲氧基-5-甲基苯肼甲基硫酸酯（1-PMS）存在下，Co^<3+>被还原为Co^<2+>，生成有色螯合物。在日本，这种方法正在作为常规大规模筛查的一种工具被引入。(5)化学合成了螺内酯[3-羟基-2-氧-1-氧-1-氧- 4 - 4 -烯]和两种新型α -酮酸[（1-环己烯基）乙醛酸酯，-（1-羟基环己烷）丙酮酸酯（烯醇形式）]的钠盐。这些化合物有望成为设计α -酮酸利用酶抑制剂的原型。事实上，其中一种被发现是一种新型的兔肌乳酸脱氢酶抑制剂。

We discovered for the first time that Bacillus sp.and Sporosarcina sp.produces phenylalanine dehydrogenase. We started our collaboration with Professor Arthur Copper of Cornell University Medical College, New York, U.S.A.We got the following results in this two years collaboration with him.(1)We have clarified the enzymological properties of phenylalanine dehydrogenase from Bacillus shaericus, B.badius, and Sporosarcina ureae. We synthesized various optically pure L-amino acids from prochiral alpha-keto acids, using the enzyme in combination with Candida formate dehydrogenase. The enzyme was effective for the reductive amination of various alpha-Keto acids. We have been successful in the synthesis of natural and unnatural L-amino acids, including L-homophenylalanine.(2)We have been successful in the synthesis of ^<13>N-Labelled L-phenylalanine, L-tyrosine, and L-DOPA.(3)We have cloned the genes for the enzymes and sequenced some of the genes. They showed similarities with leucine dehydrogenase, Streptomyces phenylalanine dehydrogenase, and glutamic acid dehydrogenase.(4)In the microplate method of PKU screening using the enzyme, L-phe is oxidized with the enzyme to phenylpyruvate, reducing NAD^+ to NADH.In the presence of NADH and the electron carrier 1-methoxy-5-methylphenazium methylsulfate (1-PMS), Co^<3+> is reduced to Co^<2+> which produces a colored chelate. The method is being introduced as a tool of the routine mass screening in Japan.(5)A spirolactone [3-hydroxy-2-oxo-1-oxaspiro [4.5] -dec-3-ene] and the sodium salts of two new alpha-keto acids [(1-cyclohexenyl) glyoxylate, beta- (1-hydoxycyclohexane) pyruvate (enol form)] were chemically synthesized. These compounds are expected to be useful as prototypes for the design of inhibitors of alpha-keto acid-utilizing enzymes. In fact, one of them was found to be a new type of inhibitor of rabbit muscle lactate dehydrogenase.