The novel substrate recognition mechanism utilized by thermophilic aspartate aminotransferase

一般认为，酶对底物具有专一性，但转氨酶是一种独特的酶，能与不同的底物结合，有两种不同的结合口袋，分别与酸性和疏水性底物结合。（每种底物的催化残留物相同。）酸性底物结合刚性区（川口et al.（1997）J.Biochem.122，55-63）和疏水底物结合柔性区（川口和Kuramitsu，（1998）J. Biol. Chem.273，18353-18364）.来自极端嗜热菌的天冬氨酸转氨酶的三维结构，根据嗜热栖热菌AspAT的X射线晶体学结构，制备了K109 V突变体，用瓦尔取代K109后，对酸性底物的酶活性降低，但对中性底物丙氨酸的酶活性显著提高，表明K109是影响嗜热厌氧杆菌AspAT酸性底物特异性的主要因素。与中性底物相互作用的动力学分析表明，嗜热嗜热菌AspAT受空间位阻较小，其底物结合口袋具有比大肠杆菌AspAT更灵活的构象，其刚性分子中的柔性活性位点可能解释了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.