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富马酸酶的结构功能特征，对其进行了酶学和遗传学研究，得到了以下结果。1)用n -乙基马来酰亚胺（NEM）对大肠杆菌天冬氨酸酶进行化学修饰失活，在不同比例的酶存在下用4 M胍-盐酸（GuHC1）使其变性。然后用pH 7.4的稀磷酸盐缓冲液稀释变性后的混合物。杂交酶显示出与未修饰亚基含量成正比的天冬氨酸酶活性，这表明只要四级结构合适，即使一些亚基是无活性的，也可以显示出酶活性。2)利用人工合成的寡核苷酸基因将参与催化活性活化的天冬氨酸酶半胱氨酸430转化为色氨酸。在酸性条件下，含trp -430的天冬氨酸酶活性提高了4倍，对二价金属离子的需要量也增加。3)现有证据表明大肠杆菌含有三种富马酶，即富马酶、富马酶和富马酶。我们纯化了一种富马酸酶，它的活性需要二价铁离子。纯化后的酶含有类似铁s簇的乌头酸酶，经鉴定为乌头酸的产物。不需要亚铁离子的FUMC也被纯化到均质。4)以前我们利用天冬氨酸β -半醛（ASA）作为天冬氨酸酶的自杀底物。为了进一步扩展本研究，我们建立了一种快速纯化酵母同型丝氨酸脱氢酶的方法，使ASA的大量供应成为可能。

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.