Tsai May, Koo Jason, Howell P Lynne
Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada.
Biochemistry. 2005 Jun 28;44(25):9034-44. doi: 10.1021/bi050346s.
Delta-crystallin, the major soluble protein component in the avian eye lens, is homologous to argininosuccinate lyase (ASL). Two delta-crystallin isoforms exist in ducks, delta1- and delta2-crystallin, which are 94% identical in amino acid sequence. While duck delta2-crystallin (ddeltac2) has maintained ASL activity, evolution has rendered duck delta1-crystallin (ddeltac1) enzymatically inactive. Previous attempts to regenerate ASL activity in ddeltac1 by mutating the residues in the 20s (residues 22-31) and 70s (residues 74-89) loops to those found in ddeltac2 resulted in a double loop mutant (DLM) which was enzymatically inactive (Tsai, M. et al. (2004) Biochemistry 43, 11672-82). This result suggested that one or more of the remaining five amino acid substitutions in domain 1 of the DLM contributes to the loss of ASL activity in ddeltac1. In the current study, residues Met-9, Val-14, Ala-41, Ile-43, and Glu-115 were targeted for mutagenesis, either alone or in combination, to the residues found in ddeltac2. ASL activity was recovered in the DLM by changing Met-9 to Trp, and this activity is further potentiated in the DLM-M9W mutant when Glu-115 is changed to Asp. The roles of Trp-9 and Asp-115 were further investigated by site-directed mutagenesis in wild-type ddeltac2. Changing the identity of either Trp-9 or Asp-115 in ddeltac2 resulted in a dramatic drop in enzymatic activity. The loss of activity in Trp-9 mutants indicates a preference for an aromatic residue at this position. Truncation mutants of ddeltac2 in which the first 8, 9, or 14 N-terminal residues were removed displayed either decreased or no ASL activity, suggesting residues 1-14 are crucial for enzymatic activity in ddeltac2. Our kinetic studies combined with available structural data suggest that the N-terminal arm in ASL/delta2-crystallin is involved in stabilizing regions of the protein involved in substrate binding and catalysis, and in completely sequestering the substrate from the solvent.
δ-晶体蛋白是鸟类晶状体中主要的可溶性蛋白质成分，与精氨琥珀酸裂解酶（ASL）同源。鸭体内存在两种δ-晶体蛋白亚型，即δ1-晶体蛋白和δ2-晶体蛋白，它们的氨基酸序列有94%的同源性。虽然鸭δ2-晶体蛋白（ddeltac2）保留了ASL活性，但进化过程使鸭δ1-晶体蛋白（ddeltac1）失去了酶活性。此前曾尝试通过将20s环（第22 - 31位氨基酸残基）和70s环（第74 - 89位氨基酸残基）中的氨基酸残基突变为ddeltac2中的相应残基来恢复ddeltac1的ASL活性，结果得到了一个双环突变体（DLM），该突变体无酶活性（蔡，M.等人（2004年）《生物化学》43卷，11672 - 11682页）。这一结果表明，DLM结构域1中其余五个氨基酸取代中的一个或多个导致了ddeltac1中ASL活性的丧失。在当前研究中，将第9位的甲硫氨酸、第14位的缬氨酸、第41位的丙氨酸、第43位的异亮氨酸和第115位的谷氨酸单独或组合地突变为ddeltac2中的相应残基。通过将第9位的甲硫氨酸变为色氨酸，DLM恢复了ASL活性，当第115位的谷氨酸变为天冬氨酸时，DLM - M9W突变体的这种活性进一步增强。通过对野生型ddeltac2进行定点诱变进一步研究了第9位色氨酸和第115位天冬氨酸的作用。改变ddeltac2中第9位色氨酸或第115位天冬氨酸的同一性会导致酶活性急剧下降。第9位色氨酸突变体活性的丧失表明该位置偏好芳香族残基。去除了前8、9或14个N端残基的ddeltac2截短突变体显示出酶活性降低或无活性，这表明第1 - 14位残基对ddeltac2的酶活性至关重要。我们的动力学研究结合现有的结构数据表明，ASL/δ2-晶体蛋白中的N端臂参与稳定蛋白质中与底物结合和催化相关的区域，并将底物完全与溶剂隔离。
