Functional implications of disulfide bond, Cys45-Cys50, in recombinant prochymosin

Bovine prochymosin (chymosin) contains three disulfide bonds: Cys45-Cys50, Cys206-Cys210 and Cys250-Cys283 (pepsin numbering). We have demonstrated that Cys250-Cys283 is not intimately involved in the catalytic mechanism of chymosin but is required for the correct refolding of prochymosin. To elucidate the functional implications of another disulfide bond of Cys45-Cys50, these two cysteine residues were replaced separately or simultaneously by site-directed mutagenesis. Like the wild-type prochymosin all the seven mutants generated (C45A, C50A, C45A/C50A, C45D, C50S, C45D/C50S, C45A/C50S) exhibit the activity of autocatalytic activation after refolding, indicating that Cys45-Cys50 is dispensable in prochymosin refolding. Spectroscopic analyses and urea-induced denaturation studies of prochymosin and four mutants tested (C45A, C50A, C45A/C50A, C45D/C50S) show that: (1) they share similar far-UV CD spectra and similar fluorescence emission spectra; (2) mutation results in a perturbance of tryptophan environment and somewhat destabilization of prochymosin conformation. However, quenching studies reveal that the only one tryptophan residue inaccessible to acrylamide is still buried in the mutated molecules. All these results suggest that the overall conformation of prochymosin is maintained after mutation. As for the enzymatic properties of pseudochymosin, the activation product of prochymosin, it has been found that elimination of Cys45-Cys50 causes a marked drop of thermostability and an alteration of substrate specificity.