Probing the Substrate Specificities of Two Enzymes of Oxidative Protein Folding

Probing the substrate specificities of two enzymes of oxidative protein folding Loading... Date Authors Journal Title Journal ISSN Volume Title Publisher University of Delaware Abstract This work probes the substrate tolerances of members of the quiescin sulfhydryl oxidase (QSOX) family of flavoenzymes and of protein disulfide isomerase (PDI) to advance our understanding of the functions of these catalysts of oxidative protein folding. Conceptually, oxidative protein folding occurs in two steps; here, the initial oxidation is promoted by QSOX, leaving reduced PDI to correct mispairings introduced by the oxidase. ☐ Part I of this dissertation explores the substrate specificity of disulfide generating enzymes to provide enzymological context for investigation of these catalysts of oxidative protein folding. QSOX enzymes are generally unable to form disulfide bonds within well-structured proteins. The use of a temperature-sensitive mutant of ubiquitin-conjugating enzyme 4 (Ubc4') as a model substrate shows that QSOX activity correlates with the unfolding of Ubc4' as monitored by circular dichroism. The fusion of Ubc4' with the more stable glutathione S-transferase domain demonstrates that QSOX can selectively introduce disulfides into the less stable domain of the fusion protein. In terms of intermolecular disulfide bond generation, QSOX is unable to crosslink well-folded globular proteins via their surface thiols. However, the utilization of a septuple mutant of ribonuclease A, which retains a single cysteine residue, demonstrates that flexible protein monomers can be directly coupled by the oxidase. These aspects of protein substrate discrimination by QSOX family members are rationalized in terms of the stringent steric requirements for disulfide exchange reactions. Furthermore, steady- and pre-steady state kinetic experiments, combined with static fluorescence approaches, indicate that while QSOX is an efficient catalyst for disulfide bond formation between mobile elements of structure, it does not appear to have a significant binding site for unfolded proteins. ☐ The later part of this dissertation focuses on the correction of mispaired disulfides. This work primarily utilizes redox trapping assays and ratiometric mass spectrometry to probe differences between the redox behaviors of QSOX and PDI. Both enzymes house an N-terminal thioredoxin domain containing a redox active Cys-x-x-Cys motif, which serves as the entry point for reducing equivalents. However, the enzymes carry out different functions. This work reveals that PDI forms a more stable mixed disulfide than QSOX with the model substrate. These results are rationalized in terms of the "hit-and-run" catalytic mechanism of the oxidase and the need for extended interactions between PDI and its mispaired client substrates.