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

Studies on the thermostabilities of the enzymes and the structures of the chimeric enzymes produced by the gene technology

酶的热稳定性及基因技术嵌合酶的结构研究

基本信息

批准号：
01440090
负责人：
TANAKA Nobuo
金额：
$ 16万
依托单位：
Tokyo Institute of Technology
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (A)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1992
项目状态：
已结题

项目摘要

Chimeric 3-isopropylmalate dehydrogenase were expressed from the fused gene between the thermophilic and mesophilic bacteria. When we compared the thermostabililty of 4M6T, the 40% of N-terminus from mesophyll and the remaining 60% from thermophile, and 2T2M6T, the 20-40% of the N-terminal residues from mesophyll, the latter was more sensitive to the heat than the former, inspire of the less numbers of residues from thermophile. The other experiments were done to recover the thermostability of the enzyme through the replacement of a residue either by the site-directed mutagenesis or induced mutagenesis by heat. We found I93L-2T2M6T and S82R-2T2M6T as thermostable enzymes.With the present project, the structural studies of these molecules have been undertaken by the X-ray crystallography. The structures were refined within 2.1A^^ﾟ resolution by PROLSQ until the conventional R factors of 0.18-0.20. Each molecule was composed of two identical subunits each of which has 345 amino acid resi … More dues. The polypeptide chain of the subunit was folded into two domains, designated first(1-99 and 252-345) and second (100-251) domains, each of which has a topologically alpha/beta structure with the other. The long arm from 140 to 150 played roles for making a dimer by the hydrogen bonds between them. The structures of chimeric enzymes differs little from thermophile. The rms deviations of the Ca atoms were 0.3A^^ﾟ for 4M6T and 0.4A^^ﾟ for 2T2M6T, respectively. The large deviations common to the two chimeras were found the loop around the 80. This movements may have something to do with the thermostability. The mutant I93L-2T2M6T released the stress between the isoleucine residue and the peptide chain by replacing it to leucine. In case of S82R-2T2M6T, the extra hydrogen bond between replaced arginine and Glu89 via water molecule. This hydrogen bond may fix the chain folding of the loop, and improve its thermostability. Many factors like entropy and enthalpy have to be taken into consideration to understand the stability of the enzyme. Less

从嗜热菌和中温菌的融合基因表达嵌合3-异丙基苹果酸脱氢酶。比较4 M6 T和2 T2 M6 T的热稳定性，发现2 T2 M6 T的热稳定性比4 M6 T的热稳定性更好，这是由于2 T2 M6 T的热稳定性比4 M6 T的热稳定性更好，2 T2 M6 T的热稳定性比4 M6 T的热稳定性更好。通过定点突变或热诱导突变取代酶的残基来恢复酶的热稳定性。我们发现I93 L-2 T2 M6 T和S82 R-2 T2 M6 T是两种热稳定酶，本项目利用X射线晶体学方法对这两种酶的结构进行了研究。通过PROLSQ在2.1埃的分辨率范围内对结构进行精细化，直到常规R因子为0.18-0.20。每个分子由两个相同的亚基组成，每个亚基有345个氨基酸残基， ...更多信息会费。亚基的多肽链折叠成两个结构域，命名为第一（1-99和252-345）和第二（100-251）结构域，其中每个结构域与另一个结构域具有拓扑α/β结构。140 ~ 150的长臂通过氢键作用形成二聚体。嵌合酶的结构与嗜热菌几乎没有区别。4 M6 T和2 T2 M6 T的Ca原子的均方根偏差分别为0.3A^^m2和0.4A^^m2。两个嵌合体共同的大偏差被发现在80周围的环。这种移动可能与热稳定性有关。突变体I93 L-2 T2 M6 T通过将异亮氨酸残基替换为亮氨酸来释放异亮氨酸残基与肽链之间的应力。在S82 R-2 T2 M6 T的情况下，精氨酸和Glu 89之间的额外氢键通过水分子被取代。这种氢键可以固定环的链折叠，并提高其热稳定性。要了解酶的稳定性，必须考虑熵和焓等许多因素。少