Recovery of argininosuccinate lyase activity in duck delta1 crystallin.
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.
Tsai
May
M
Structural Biology and Biochemistry, Research Institute, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada.
Koo
Jason
J
Howell
P Lynne
PL
D000818:Animals; D001123:Argininosuccinate Lyase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004372:Ducks; D004795:Enzyme Stability; D019143:Evolution, Molecular; D006801:Humans; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D038207:delta-Crystallins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D038207:delta-Crystallins; D001123:Argininosuccinate Lyase