Sampaleanu Liliana M, Yu Bomina, Howell P Lynne
Structural Biology and Biochemistry Program, Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
J Biol Chem. 2002 Feb 8;277(6):4166-75. doi: 10.1074/jbc.M107465200. Epub 2001 Nov 6.
The major soluble avian eye lens protein, delta crystallin, is highly homologous to the housekeeping enzyme argininosuccinate lyase (ASL). ASL is part of the urea and arginine-citrulline cycles and catalyzes the reversible breakdown of argininosuccinate to arginine and fumarate. In duck lenses, there are two delta crystallin isoforms that are 94% identical in amino acid sequence. Only the delta2 isoform has maintained ASL activity and has been used to investigate the enzymatic mechanism of ASL. The role of the active site residues Ser-29, Asp-33, Asp-89, Asn-116, Thr-161, His-162, Arg-238, Thr-281, Ser-283, Asn-291, Asp-293, Glu-296, Lys-325, Asp-330, and Lys-331 have been investigated by site-directed mutagenesis, and the structure of the inactive duck delta2 crystallin (ddeltac2) mutant S283A with bound argininosuccinate was determined at 1.96 A resolution. The S283A mutation does not interfere with substrate binding, because the 280's loop (residues 270-290) is in the open conformation and Ala-283 is more than 7 A from the substrate. The substrate is bound in a different conformation to that observed previously indicating a large degree of conformational flexibility in the fumarate moiety when the 280's loop is in the open conformation. The structure of the S283A ddeltac2 mutant and mutagenesis results reveal that a complex network of interactions of both protein residues and water molecules are involved in substrate binding and specificity. Small changes even to residues not involved directly in anchoring the argininosuccinate have a significant effect on catalysis. The results suggest that either His-162 or Thr-161 are responsible for proton abstraction and reinforce the putative role of Ser-283 as the catalytic acid, although we cannot eliminate the possibility that arginine is released in an uncharged form, with the solvent providing the required proton. A detailed enzymatic mechanism of ASL/ddeltac2 is presented.
主要的可溶性鸟类晶状体蛋白δ-晶体蛋白与管家酶精氨琥珀酸裂解酶（ASL）高度同源。ASL是尿素和精氨酸-瓜氨酸循环的一部分，催化精氨琥珀酸可逆地分解为精氨酸和富马酸。在鸭晶状体中，有两种δ-晶体蛋白异构体，其氨基酸序列的同源性为94%。只有δ2异构体保留了ASL活性，并已用于研究ASL的酶促机制。通过定点诱变研究了活性位点残基Ser-29、Asp-33、Asp-89、Asn-116、Thr-161、His-162、Arg-238、Thr-281、Ser-283、Asn-291、Asp-293、Glu-296、Lys-325、Asp-330和Lys-331的作用，并且以1.96 Å的分辨率确定了结合精氨琥珀酸的无活性鸭δ2晶体蛋白（ddeltac2）突变体S283A的结构。S283A突变不干扰底物结合，因为280环（残基270-290）处于开放构象，且Ala-283与底物的距离超过7 Å。底物以与先前观察到的不同构象结合，这表明当280环处于开放构象时，富马酸部分具有很大程度的构象灵活性。S283A ddeltac2突变体的结构和诱变结果表明，蛋白质残基和水分子的复杂相互作用网络参与了底物结合和特异性。即使对不直接参与锚定精氨琥珀酸的残基进行微小改变，也会对催化产生显著影响。结果表明，His-162或Thr-161负责质子提取，并强化了Ser-283作为催化酸的假定作用，尽管我们不能排除精氨酸以不带电荷的形式释放，溶剂提供所需质子的可能性。本文提出了ASL/ddeltac2详细的酶促机制。
