The novel substrate recognition mechanism utilized by thermophilic aspartate aminotransferase

人们一直认为，一种酶对其底物有很强的专一性。但氨基转移酶是一种非常独特的酶，它可以结合不同的底物。这些酶对酸性和疏水性底物有两种不同类型的结合口袋。(每种底物的催化残留物是相同的。)酸性底物结合硬质区域(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)。化学。用X射线结晶学方法测定了极端嗜热菌Hb8的天冬氨酸转氨酶的三维结构。根据嗜热菌Hb8的X射线晶体结构，制备了K109V突变株。用Val替换K109导致对酸性底物失去活性，但对中性底物丙氨酸的活性增加。这些结果表明，K109是决定嗜热菌对酸性底物专一性的主要决定因素。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.coliAspat具有更灵活的构象，刚性T.thermophilus Aspat分子中灵活的活性中心可能是其即使在室温下也具有高活性的原因。

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.