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，酶对其底物是非常特异的。但是氨基转移酶是非常独特的酶，可以结合底物的结合完全不同的酶。这些酶具有两种不同类型的结合口袋，用于酸性和疏水性底物。 (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 T.thermophilus aspats的酸性底物特异性的决定因素。对与中性底物相互作用的相互作用的动力学分析表明，T.thermophilus aspat受到较小的空间障碍，其底物结合口袋的构象比E.Coli ASPAT更灵活。在刚性T.thermid t.thermophilus aspat Molecule中，柔性活性位点甚至可以在室温下解释其高活性。