Studies on structure-function relationship of enzymes catalyzing asymmetric syntheses of Michael addition type.

为了阐明催化Michael加成型不对称合成反应的天冬氨酸酶和天冬氨酸酶的结构-功能特征，我们进行了酶学和遗传学研究，得到了以下结果：1）大肠杆菌天冬氨酸酶，由4个相同的亚基组成，通过用N-乙基马来酰亚胺（NEM）化学修饰失活，并在4 M盐酸胍（GuHCl）中变性，在不同比例的活性酶存在下。然后通过用稀磷酸盐缓冲液（pH 7.4）稀释使变性的混合物复性。结果表明，该酶的活性与未修饰亚基的含量成正比，表明只要四级结构合适，即使某些亚基失活，该酶也能表现出活性。2）利用人工合成的寡核苷酸将参与该酶催化活性激活的半胱氨酸430基因转化为色氨酸。Trp-430酶活在酸性条件下提高了4倍，对二价金属离子的需求量也增加。大肠杆菌含有三种酶，即FUMA、FUMB和FUMC。我们纯化了一种需要二价亚铁离子才能发挥活性的酶。纯化的酶中含有铁-S簇样顺乌头酸酶，经鉴定为fumA的产物。4）我们利用天冬氨酸β-半醛（阿萨）作为天冬氨酸酶的自杀底物。为了进一步研究，我们建立了酵母高丝氨酸脱氢酶的快速纯化方法，使大量生产阿萨成为可能。

In order to elucidate the structure-function characteristics of aspartases and fumarases, which catalyze asymmetric synthetic reactions of Michael addition type, enzymatic and genetic studies were carried out, and the following results were obtained.1) Aspartase of Escherichiac coli, composed of 4-identical subunits was inactivated by chemical-modification with N-ethylmaleimide (NEM) and denatured in 4 M guanidine-HCl (GuHC1) in the presence of various ratio of the active enzyme. The denatured mixture was then renatured by dilution with dilute phosphate buffer, pH 7.4. The hybridized enzyxne exhibited the aspartase activity in proportion to the content of the unmodified subunit, indicating that as long as the quaternary structure is proper, the enzyme activity can be exhibited, even if some subunits are inactive.2) Cysteind 430 of aspartase, which participates in the activation of the catalytic activity was genetically converted to tryptophan using synthetic oligonucleotides. Trp-430-containing aspartase exhibited 4 times higher enzyme activity at acidic pH, and the requirement for divalent metal ions was also increased.3) Available-evidence suggests that E. colicells contain three fumarases, FUMA, FUMB, dnd FUMC. We purified a fumarase which required divalent ferrous ions for its activity. The purified enzyme was found to contain iron-S cluster like aconitase and was identified as a product of fumA. FUMC, which did not require ferrous ions was also purified to homogeneety.4) Freviously we utilized aspartic beta-semialdehyde (ASA) as a kind of suicide substrate of aspartase. In order to extend this study, we established a rapid purification procedure for homoserine dehydrogenase of yeast, by which a large amount of ASA supply became possible.