Loss of function mutations in conserved regions of the human arginase I gene.

We have utilized SSCP analysis to identify disease-causing mutations in a cohort with arginase deficiency. Each of the patient's mutations was reconstructed in vitro by site-directed mutagenesis to determine the effect of the mutations on enzyme activity. In addition we identified six areas of cross-species homology in the arginase protein, four containing conserved histidine residues thought to be important to Mn(2+)-dependent enzyme function. Mapping patient mutations in relationship to the conserved regions indicates that substitution mutations within the conserved regions and randomly occurring microdeletions and nonsense mutations have a significant effect on enzymatic function. In vitro mutagenesis was utilized to create nonpatient substitution mutations in the conserved histidine residues to verify their importance to arginase activity. As expected, replacement of histidine residues with other amino acids dramatically reduces arginase activity levels in our bacterial expression system.