Using [delta]1 and [delta]2 crystallin to investigate the enzymatic mechanism of argininosuccinate lyase

Argininosuccinale lyase (ASL) catalyses the reversible breakdown of argininosuccinate to arginine and fumarate, a reaction involved in arginine biosynthesis, and in the urea and arginine-citrulline cycles. During evolution, ASL became highly expressed in the avian eye lens as δ crystallin. In the duck eye lens ASL recruitment was followed by gene duplication, resulting in two δ crystallin proteins. Although the two isoforms are 94% identical in amino acid sequence, only δ2 crystallin (dδc2) is the duck orthologue of ASL while δ1 crystallin (dδc1) is enzymatically inactive. In this thesis, structural and functional analysis of wild type and site-directed mutants of dδc1 and dδc2 have been used to investigate the enzymatic mechanism of argininosuccinate lyase. Domain swapping experiments were used to assess the role of each structural domain of the δ crystallin monomer. The kinetic and thermodynamic analysis of the chimeras revealed that only the first, N-terminal domain (domain 1) is crucial for the recovery of enzymatic activity in the inactive dδc1. As the residues implicated in the catalytic mechanism of dδc2/ASL are conserved in dδc1, we postulate that amino-acid substitutions in domain 1 of dδc1 are critical for substrate binding and hence catalysis. The crystal structures of wild type dδc1 and dδc2 crystallin have been solved and compared with the available δ crystallin structures. These structural comparisons indicate that both intra- and inter-species conformational changes occur in two loop regions of domain 1 (residues 23–32 and 74–89). The structure of dδc1 revealed the presence of a sulfate anion in the active site region that may mimic the fumarate moiety of the argininosuccinate substrate. This anion induced a large conformational change in the 280's loop and a rigid body motion in the C-terminal domain (domain 3). The results suggest that S281 may play the role of the acid catalyst in the enzymatic mechanism of dδc2/ASL. The proposed role of the 23–32 and 74–89 loops of domain 1 in substrate binding was investigated by mutagenesis studies. Single and multiple mutants of dδc1 were generated but no recovery of ASL activity was observed, suggesting that although the two loops might play a significant role for the enzyme function, the other substitutions in domain 1 are also important. In this context, the possible catalytic role of the N-terminal tail (residues 1–18) is discussed. The structure determination of the inactive S281A dδc2 mutant with argininosuccinate bound to all four active sites has reinforced the hypothesis that S281 plays a catalytic rather than a substrate-binding role. Extensive mutagenesis mapping of the active site region was performed and an extensive network of interactions was shown to be involved in substrate binding and catalysis. Combined, the above results have enabled us to propose a detailed catalytic mechanism for dδc2/ASL.

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