Chakraborty A R, Davidson A, Howell P L
Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
Biochemistry. 1999 Feb 23;38(8):2435-43. doi: 10.1021/bi982150g.
Delta-crystallins are the major structural eye lens proteins of most birds and reptiles and are direct homologues of the urea cycle enzyme argininosuccinate lyase. There are two isoforms of delta-crystallin, delta Iota and delta IotaIota, but only delta IotaIota crystallin exhibits argininosuccinate lyase (ASL) activity. At the onset of this study, the structure of argininosuccinate lyase/delta IotaIota crystallin with bound inhibitor or substrate analogue was not available. Biochemical and X-ray crystallographic studies had suggested that H162 may function as the catalytic base in the argininosuccinate lyase/delta IotaIota crystallin reaction mechanism, either directly or indirectly through the activation of a water molecule. The identity of the catalytic acid was unknown. In this study, the argininosuccinate substrate was modeled into the active site of duck delta IotaIota crystallin, using the coordinates of an inhibitor-bound Escherichia coli fumarase C structure to orient the fumarate moiety of the substrate. The model served as a means of identifying active site residues which are positioned to potentially participate in substrate binding and/or catalysis. On the basis of the results of the modeling, site-directed mutagenesis was performed on several amino acids, and the kinetic and thermodynamic properties of each mutant were determined. Kinetic studies reveal that five residues, R115, N116, T161, S283, and E296, are essential for catalytic activity. Determination of the free energy of unfolding/refolding of wild-type and mutant delta II crystallins revealed that all constructs exhibit similar thermodynamic stabilities. During the course of this work, the structure of an inactive delta IotaIota crystallin mutant with bound substrate was solved [Vallee et al. (1999) Biochemistry 38, 2425-2434], which has allowed the kinetic data to be interpreted on a structural basis.
δ-晶体蛋白是大多数鸟类和爬行动物晶状体的主要结构蛋白，是尿素循环酶精氨琥珀酸裂解酶的直接同源物。δ-晶体蛋白有两种同工型，即δⅠ和δⅡ，但只有δⅡ晶体蛋白具有精氨琥珀酸裂解酶（ASL）活性。在本研究开始时，精氨琥珀酸裂解酶/δⅡ晶体蛋白与结合抑制剂或底物类似物的结构尚未可知。生化和X射线晶体学研究表明，H162可能在精氨琥珀酸裂解酶/δⅡ晶体蛋白反应机制中作为催化碱，要么直接作用，要么通过激活水分子间接作用。催化酸的身份未知。在本研究中，利用结合抑制剂的大肠杆菌延胡索酸酶C结构的坐标将精氨琥珀酸底物模拟到鸭δⅡ晶体蛋白的活性位点中，以使底物的延胡索酸部分定向。该模型作为一种识别可能参与底物结合和/或催化的活性位点残基的方法。基于模拟结果，对几个氨基酸进行了定点诱变，并测定了每个突变体的动力学和热力学性质。动力学研究表明，五个残基R115、N116、T161、S283和E296对催化活性至关重要。野生型和突变型δⅡ晶体蛋白解折叠/重折叠自由能的测定表明，所有构建体都表现出相似的热力学稳定性。在这项工作过程中，解析了结合底物的无活性δⅡ晶体蛋白突变体的结构[瓦利等人（1999年）《生物化学》38卷，2425 - 2434页]，这使得能够在结构基础上解释动力学数据。
