LOSS OF FUNCTION MUTATIONS IN CONSERVED REGIONS OF THE HUMAN ARGINASE-I GENE

We have utilized SSCP analysis to identify disease-causing mutations i n a cohort with arginase deficiency. Each of the patient's mutations w as reconstructed in vitro by site-directed mutagenesis to determine th e effect of the mutations on enzyme activity. In addition we identifie d six areas of cross-species homology in the arginase protein, four co ntaining conserved histidine residues thought to be important to Mn2+- dependent enzyme function. Mapping patient mutations in relationship t o the conserved regions indicates that substitution mutations within t he conserved regions and randomly occurring microdeletions and nonsens e mutations have a significant effect on enzymatic function. In vitro mutagenesis was utilized to create nonpatient substitution mutations i n the conserved histidine residues to verify their importance to argin ase activity. As expected replacement of histidine residues with other amino acids dramatically reduces arginase activity levels in our bact erial expression system. (C) 1996 Academic Press, Inc.