HUMAN SULFITE OXIDASE R160Q - IDENTIFICATION OF THE MUTATION IN A SULFITE OXIDASE-DEFICIENT PATIENT AND EXPRESSION AND CHARACTERIZATION OF THE MUTANT ENZYME

Sulfite oxidase catalyzes the terminal reaction in the degradation of sulfur amino acids. Genetic deficiency of sulfite oxidase results in n eurological abnormalities and often leads to death at an early age. Th e mutation in the sulfite oxidase gene responsible for sulfite oxidase deficiency in a 5-year-old girl was identified by sequence analysis o f cDNA obtained from fibroblast mRNA to be a guanine to adenine transi tion at nucleotide 479 resulting in the amino acid substitution of Arg -160 to Gin, Recombinant protein containing the R160Q mutation was exp ressed in Escherichia coli, purified, and characterized. The mutant pr otein contained its full complement of molybdenum and heme, but exhibi ted 2% of native activity under standard assay conditions. Absorption spectroscopy of the isolated molybdenum domains of native sulfite oxid ase and of the R160Q mutant showed significant differences in the 480- and 350-nm absorption bands, suggestive of altered geometry at the mol ybdenum center. Kinetic analysis of the R160Q protein showed an increa se in K-m for sulfite combined with a decrease in k(cat) resulting in a decrease of nearly 1,000-fold in the apparent second-order rate cons tant k(cat)/K-m. Kinetic parameters for the in vitro generated R160K m utant were found to be intermediate in value between those of the nati ve protein and the R160Q mutant. Native sulfite oxidase was rapidly in activated by phenylglyoxal, yielding a modified protein with kinetic p arameters mimicking those of the R160Q mutant. It is proposed that Arg -160 attracts the anionic substrate sulfite to the binding site near t he molybdenum.