Mutational Analysis of Duck δ2 Crystallin and the Structure of an Inactive Mutant with Bound Substrate Provide Insight into the Enzymatic Mechanism of Argininosuccinate Lyase

Abstract

The major soluble avian eye lens protein, ␦ 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 ␦ crystallin isoforms that are 94% identical in amino acid sequence. Only the ␦2 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 ␦2 crystallin (d␦c2) mutant S283A with bound argininosuccinate was determined at 1.96 Å 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 Å 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 d␦c2 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/d␦c2 is presented.␦ Crystallin is a taxon-specific crystallin that represents the major soluble protein in the eye lenses of birds and terrestrial reptiles. This crystallin was recruited from the housekeeping enzyme argininosuccinate lyase (ASL 1 ) (1, 2) through a process called gene sharing (3,4). ASL is a cytosolic enzyme that catalyzes the reversible breakdown of argininosuccinate to arginine and fumarate. Gene recruitment of ASL was followed by gene duplication and resulted in two proteins, ␦1 and ␦2 crystallin. In ducks, there is 94% amino acid sequence identity between the ␦1 and ␦2 isoforms (d␦c1 and d␦c2), and 69 and 71% identity to human ASL, respectively (2, 5). During evolution, the ␦1 isoform has presumably become a more specialized lens protein and has lost ASL activity (6 -8), while the ␦2 isoform has retained enzymatic activity and is the duck orthologue of ASL in non-lens tissues (9, 10).ASL and ␦ crystallin are members of a superfamily of enzymes that are active as homotetramers and catalyze similar ␤-elimination reactions in which a C ␣ -N or C ␣ -O bond is cleaved with the subsequent release of fumarate as one of the pro...

Disruption of a Salt Bridge Dramatically Accelerates Subunit Exchange in Duck δ2 Crystallin

“…All homomutant proteins were inactive, except for ␦2-N114D-His, which had 0.2 Ϯ 0.1% wt␦2-His activity (25). Coexpression of ␦2-D87N-His with ␦2-N114D-His yielded tetramers with 0.2 Ϯ 0.1% wt␦2-His activity.…”

“…The in vitro activity was measured spectrophotometrically by monitoring the increase of fumarate at 240 nm (⑀ ϭ 2.44 mM Ϫ1 cm Ϫ1 ) as described by Sampaleanu et al (25). Briefly, the substrate, sodium argininosuccinate (Sigma), was mixed with 10 -20 g of enzyme in reaction buffer (60 mM Tris-HCl, pH 7.5) at final concentrations ranging from 0.02 to 2.0 mM.…”

“…Expression Vectors-The construction of pET-3d vectors expressing wild-type duck ␦2 crystallin and the D87N, N114D, T159D, and S281T single point mutants with a C-terminal His 6 tag was described previously (25). For the expression of ␦2 crystallin heterotetramers, vectors containing the full-length cDNA and T7 promoter regions for the N114D, T159D, or S281T mutant with the full-length cDNA and T7 promoter region for the D87N mutant were created.…”

“…The catalytic activity of the ␦2 crystallin active site homomutant proteins has been characterized previously (25). All homomutant proteins were inactive, except for ␦2-N114D-His, which had 0.2 Ϯ 0.1% wt␦2-His activity (25).…”

“…␦2 Crystallin is more thermostable than ASL (23) making it more amenable to in vitro analysis. A large library of duck ␦2 crystallin mutants also exists (24,25) which can be used to study complementation.…”