Ogawa H, Fujioka M
J Biol Chem. 1978 May 25;253(10):3666-70.
Saccharopine dehydrogenase (N6-(glutar-2-yl)-L-ly-sine:NAD oxidoreductase (L-lysine-forming)) from baker's yeast was purified to homogenicity. The overall purification was about 1,200-fold over the crude extract with a yield of about 24%. The purified enzyme had a sedimentation coefficient (S20,w) of 3.0 S. The molecular weight determinations by sedimentation equilibrium, Sephadex G-100 gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a value of about 39,000 and, therefore, saccharopine dehydrogenase is a single polypeptide chain enzyme. A Stokes radius of 27 A and a diffusion constant of 7.9 X 10(-7) cm2 s-1 were obtained from Sephadex gel filtration chromatography. The enzyme had a high isoelectric pH of 10.1. The NH2-terminal sequence was Ala-Ala----. The enzyme possessed 3 cysteine residues/molecule; no disulfide bond was present. Incubation of saccharopine dehydrogenase with p-chloromercuribenzoate or iodoacetate resulted in complete loss of enzyme activity. Whereas the coenzyme and substrates were ineffective in protecting from inactivation by p-chloromercuribenzoate, iodoacetate inhibition was protected by excess coenzyme.
对来自面包酵母的酵母氨酸脱氢酶（N6-(戊-2-基)-L-赖氨酸：NAD氧化还原酶（形成L-赖氨酸））进行了纯化，使其达到均一性。与粗提物相比，总体纯化倍数约为1200倍，产率约为24%。纯化后的酶沉降系数（S20,w）为3.0 S。通过沉降平衡、葡聚糖G-100凝胶过滤和十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定的分子量约为39000，因此，酵母氨酸脱氢酶是一种单条多肽链酶。通过葡聚糖凝胶过滤色谱法测得斯托克斯半径为27 Å，扩散常数为7.9×10(-7) cm2 s-1。该酶的等电pH值较高，为10.1。氨基末端序列为Ala-Ala----。该酶每个分子含有3个半胱氨酸残基；不存在二硫键。用对氯汞苯甲酸或碘乙酸孵育酵母氨酸脱氢酶会导致酶活性完全丧失。虽然辅酶和底物不能保护该酶免受对氯汞苯甲酸的失活作用，但过量的辅酶可保护其免受碘乙酸的抑制。
