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The novel substrate recognition mechanism utilized by thermophilic aspartate aminotransferase

嗜热天冬氨酸转氨酶利用的新型底物识别机制

基本信息

批准号：
09680619
负责人：
KURAMITSU Seiki
金额：
$ 2.18万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

It has been believed that an enzyme is quite specific for its substrate.But aminotransferases are quite unique enzymes which can bind quite different binds of substrate.These enzymes have two different types of binding pockets for acidic and hydrophobic substrates. (Catalytic residue is identical for each substrates.) Acidic substrate binds rigid region (Kawaguchi et al.(1997) J.Biochem.122, 55-63) and hydrophobic substrate binds flexible region (Kawaguchi and Kuramitsu, (1998) J.Biol. Chem. 273, 18353-18364).The three-dimensional structure of aspartate aminotransferase from extreme thermophile, Thermus thermophilus HB8 have been determined by X-ray crystallography.In view of the X-ray crystallographic structure of T.thermophilus AspAT, K1O9V mutant was prepared.Replacing K109 with Val resulted in loss of activity toward acidic substrates, but increased that toward the neutral substrate, alanine, considerably.These results indicate that K109 is a major determinant of the acidic substrate specificity of T.thermophilus AspATs. Kinetic analysis of the interactions with neutral substrates indicated that T.thermophilus AspAT is subject to less steric hindrance and its substrate-binding pocket has a more flexible conformation than E.coli AspAT.A flexible active site in the rigid T.thermophilus AspAT molecule may explain its high activity even at room temperature.

人们认为酶对其底物具有相当的特异性。但是转氨酶是非常独特的酶，可以结合完全不同的底物结合。这些酶具有针对酸性和疏水性底物的两种不同类型的结合袋。 (每个底物的催化残基是相同的。)酸性底物结合刚性区域(Kawaguchi et al.(1997) J.Biochem.122, 55-63)，疏水底物结合柔性区域(Kawaguchi and Kuramitsu, (1998) J.Biol. Chem. 273, 18353-18364)。天冬氨酸的三维结构采用X射线晶体学方法对来自极端嗜热菌Thermus thermophilus HB8的转氨酶进行了测定。鉴于T.thermophilus AspAT的X射线晶体结构，制备了K1O9V突变体。用Val代替K109导致对酸性底物的活性丧失，但对中性底物丙氨酸的活性显着增加。这些结果表明K109是一种主要的转氨酶。 T.thermophilus AspAT 的酸性底物特异性的决定因素。与中性底物相互作用的动力学分析表明，T.thermophilus AspAT 受到的空间位阻较小，其底物结合口袋比 E.coli AspAT 具有更灵活的构象。刚性 T.thermophilus AspAT 分子中的柔性活性位点可以解释其即使在室温下也具有高活性。