Functional implications of disulfide bond, Cys206-Cys210, in recombinant prochymosin (chymosin)

Prochymosin (chymosin) contains three disulfide bonds: Cys45-Cys50, Cys206- Cys210, and Cys250-Cys283. We have demonstrated that Cys250-Cys283 is indis pensable fur correct refolding of prochymosin, whereas Cys45-Cys50 is dispe nsable but has some contribution to the stability and substrate specificity of the enzyme. Here, we report the results about the functions of Cys206-C ys210 by site-directed mutagenesis studies. In a glutathione redox system C 206A/C210A mutant exhibited oxidative refolding, kinetics and efficiency (s imilar to 40% reactivation) similar to those of the wild-type prochymosin, indicating that Cys206-Cys210 is also dispensable for refolding. However, C 206S/C210S and single-site mutants (C210A, C210S, and C206A) showed only ab out 3 and 0-0.4% reactivation, respectively. This is quite different from t he Cys45-Cys50 deficient mutants (C45A, C50A, C45A/C50A, C45D, C50S, C45D/C 50S, C45A/C50S), which have comparable refolding efficiencies, implying tha t the substituents at position 206 and 210 play more important role in dete rmining correct refolding than those at position 45 and 50. Urea-induced de naturation and fluorescence quenching studies indicated that the prochymosi n mutants C206A/C210A and C206S/C210S were 2.1 and 4.8 kJ/mol less stable t han prochymosin and some tryptophan residue in the mutated molecules was le ss exposed. However, the wild-type and mutant prochymosins shared similar f ar-UV CD and fluorescence emission spectra and similar specific potential a ctivity, suggesting that the overall conformation was maintained after muta tion. Activity assay and kinetic analysis revealed that mutation did not ch ange the specific milk-clotting activity significantly but resulted in an i ncrease in K-m, and k(cat) toward a hexapeptide substrate. On the basis of the above-mentioned perturbance of tryptophanyl microenvironment and the th ree-dimensional structure of chymosin, we proposed that deletion of Cys206- Cys210 may induce a propagated conformational change, resulting in a pertur bance of the local conformation around active-site cleft and in turn, an al teration of the substrate specificity.