Abstract 1572 BIOCHEMICAL STUDY OF MUTANTS OF HUMAN ARGINASE I, CAUSING ARGININEMIA

Background: Human arginase is a trimeric metalloprotein of 322 amino acids per monomer that catalyzes the hydrolysis of arginine to ornithine and urea in the last step of the urea cycle. The gene that encodes hepatic arginase (ARG1), located at locus 6q23 in humans, covers 11 kb including 8 exons and 7 introns , with a transcript of 1,393 bp. Another isoform has been described, a mitochondrial ARG2 encoded by another gene and which shares 60% homology and whose gene product has been found in tissues such as kidney, small intestine and brain, whose function is not completely known. Mutations in the ARG1 gene cause an autonomic recessive disorder called argininemia. The clinical manifestations of this condition range from mental and developmental delay, seizures, spastic tetraplegia and present since childhood, some in mild form and others more severe, but clearly there is no correlation between genotype and phenotype because the variation in clinical severity cannot be explained by differences in the nature of the mutations. Around 70 mutations have been described throughout this gene related to this condition, the most frequent being the so-called nonsense mutations, followed by premature arrest (nonsense), splicing, deletions, insertions and duplications mutations. Arginase deficiency is pan-ethnic and has been reported with an incidence ranging from 1:350,000 to 1:2,000,000 births in Japanese, French-Canadian and American populations. However, in the Mexican population there are only 2 reports describing mutations in patients with argininemia and the incidence of the disease is unknown. Interestingly, residue H141 is found in a highly conserved region across the phylogenetic scale, including arginase from organisms such as non-mammalian vertebrates, invertebrates, yeasts, and bacteria. On the other hand, the T134I mutation, present in the Brazilian population, has a high prevalence and in a report in patients with argininemia this mutation was found in homozygous form in 43% of the patients analyzed; However, the in silico study they did does not show that this mutation destabilizes the structure of the protein. Although only a small fraction of mutations in the ARG1 gene have been studied systematically, biochemically and enzymatically, the vast majority have been predicted in in silico studies of the impact of the mutation on the protein. Methodology: Cloning of the human ARG gene in an overexpression vector. For mutant proteins, site-directed mutagenesis will be performed. Once all the proteins have been obtained, they will be characterized and compared biochemically and structurally. Results. Both mutant proteins present kinetic differences with respect to the native one, which indicates that the change in the aforementioned residues indirectly impacts the activity of the enzyme.

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