The formation of disulfide bonds from cysteine free thiols is essential to a wide number of proteins for structure and function; however, disulfide bond formation in eukaryotes is not a well-understood process. Quiescin - sulfhydryl oxidase (QSOX) is a candidate enzyme due to its ability to rapidly catalyze the formation of disulfide bonds. All QSOX family members contain a number of conserved cysteines; among these are six residues that make up three conserved CxxC motifs (where C represents cysteine and x is any amino acid). Based upon previous work accomplished with avian QSOX and by comparison to the mechanistic model for the QSOX Erv/ALR domain homologue, yeast Erv2p, it was thought that these cysteines would be essential to catalysis. In order to directly investigate the roles of these six cysteines, a recombinant form of the enzyme is needed followed by mutation of the residues to analyze their effect on catalysis. These objectives form the basis of this work.

For this work, the human QSOX1 short form, HsQSOX1b, was used. The production of moderate yields of soluble protein was achieved and initial characterization of this enzyme resulted in kinetic parameters that correlated with those previously determined for the avian egg white and bovine milk enzymes. On the basis of this recombinant form, site-directed mutagenesis was employed to explore the effects on catalysis of the six cysteine residues contained in the three conserved CxxC motifs. This work shows that the two cysteines of the third CxxC motif were not required for catalysis. This remarkable finding resulted in a new proposal for the mechanism of QSOX catalysis that is outlined in this work.

This work also initiated investigations into QSOX inhibition. Arsenicals are well known compounds and are tight binders of vicinal dithiols, such as those contained in the CxxC motifs of QSOX. The availability of the QSOX CxxC mutants and the on-going research in our lab into arsenical compounds, led to a preliminary project on QSOX inhibition. These studies suggest that the CxxC disulfide proximal to the flavin cofactor may be the site of arsenic inhibition.