1K7W: Crystal Structure Of S283a Duck Delta 2 Crystallin Mutant

Citation:
Abstract
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.
PDB ID: 1K7WDownload
MMDB ID: 18934
PDB Deposition Date: 2001/10/22
Updated in MMDB: 2007/10
Experimental Method:
x-ray diffraction
Resolution: 1.96  Å
Source Organism:
Similar Structures:
Biological Unit for 1K7W: tetrameric; determined by author and by software (PISA)
Molecular Components in 1K7W
Label Count Molecule
Proteins (4 molecules)
4
Delta 2 Crystallin
Molecule annotation
Chemicals (4 molecules)
1
4
* Click molecule labels to explore molecular sequence information.

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