Intragenic complementation and protein oligomerization studies in argininosuccinate lyase and its homologue delta crystallin.
Published: 2004
Language: eng
Number of pages: 261
Description: Argininosuccinate lyase (ASL) is a ubiquitous enzyme that catalyzes the reversible cleavage of argininosuccinate to arginine and fumarate. This reaction is important both for the detoxification of ammonia via the urea cycle and for the biosynthesis of arginine. Through a process called 'gene sharing' ASL was recruited to the eye lens of birds and reptiles where it acts as the major lens crystallin. Both ASL and delta crystallin exist as homotetramers with a monomer molecular weight of approximately 50 kDa. Extensive intragenic complementation was observed at the ASL locus in humans. Intragenic complementation occurs when certain combinations of mutant alleles produce an enzyme with greater catalytic activity than is observed in the homozygous state of either mutant.In this thesis, ASL and delta2 crystallin were used as model systems to study intragenic complementation and protein oligomerization. The structure and function of mutant proteins possessing amino acid substitutions associated with ASL deficiency were characterized. Mutations were found to either disturb the active site or drastically destabilize the protein such that proper metabolic function would be compromised. By coexpressing different pairs of mutants, intragenic complementation was found to occur between two active site mutants by the regeneration of native-like active sites and between stable and unstable mutants due to the increase in stability upon oligomerization. Complementation was also observed between delta2 crystallin mutants and between delta2 crystallin and ASL, implying that ASL and delta2 crystallin have similar subunit interfaces and that the two proteins fold in the same manner. Both proteins were found to unfold in guanidine hydrochloride via a partially folded dimeric intermediate. Extensive site-directed mutagenesis at the subunit interface and of residues buried in the monomer suggested that unfolding occurs independent of the oligomeric state and illustrated the importance of inter-subunit salt-bridges for maintaining tetramer stability. Intragenic complementation was observed between mutant proteins with opposite amino acid substitutions in the subunit interface. Together these studies have provided insight into the pathology of argininosuccinic aciduria and the folding mechanism of ASL and delta2 crystallin, and have illustrated the value of intragenic complementation studies when examining subunit interactions in oligomeric proteins.
Notes: Advisers:  P. L. Honell; A. R. Davidson.

Thesis (Ph.D.)--University of Toronto, 2004.

Electronic version licensed for access by U. of T. users.

Source: Dissertation Abstracts International, Volume: 65-10, Section: B, page: 5140.