权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

迈克尔加成型不对称合成酶的构效关系研究。

基本信息

批准号：
63470136
负责人：
TOKUSHIGE Masanobu
金额：
$ 4.8万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

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.

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和功能特征，我们进行了酶学和遗传学研究，得到以下结果：1)用N-乙基马来酰亚胺(NEM)化学修饰由4个相同亚基组成的大肠杆菌天冬氨酸酶，并在不同的活性酶比例下在4M盐酸胍(GuHCl)中变性。变性后的混合物用稀磷酸盐缓冲液(pH 7.4)稀释复性。杂交酶的天冬氨酸酶活性与未修饰亚基的含量成正比，表明只要四级结构正确，即使某些亚基失活，也能表现出酶的活性。2)利用合成的寡核苷酸将参与催化活性激活的天冬氨酸酶的半胱氨酸430基因转化为色氨酸。含有Trp-430的天冬氨酸氨基转移酶在酸性条件下表现出高4倍的酶活力，对二价金属离子的需求也增加。3)现有证据表明，大肠杆菌含有三种延胡索酶，FUMA，FUMB，DND FUMC。我们纯化了一种富马酸酶，它的活性需要二价亚铁离子。纯化后的酶含有铁S簇状乌头酸酶，经鉴定为富马产物。对不需要亚铁离子的FUMC也进行了纯化，得到了均一的FUMC。4)采用天冬氨酸-β-半醛(ASA)作为天冬氨酸酶的自杀底物。为了扩大这一研究范围，我们建立了一种酵母高丝氨酸脱氢酶的快速纯化方法，使AsA的大量供应成为可能。