Functional implications of the 21-24 loop in recombinant prochymosin

摘要

To investigate the role of the 21-24 (pepsin numbering) loop in prochymosin, the amino acid residues GTPP at positions 21 through 24 were replaced with GG, the equivalent loop residues from its homologous protein, penicillopepsin, or SG, GS by site-directed mutagenesis. The mutants except GTPP(21-24)GS could be expressed in Escherichia coli. Activation studies indicated that the refolded prochymosin mutants were capable of undergoing autocatalytic activation to produce pseudochymosin by cleaving its N-terminal 27 amino acid residues at pH 2. The resulting pseudochymosin mutants were able to convert into chymosin at pH 5.5 by further autocatalytic cleavage to remove additional 15 amino acid residues. These results demonstrate that the prochymosin analogs can fold into an active state from an unfolded state and that the pseudochymosin analogs can proceed in the transformation from one active form into another active form. Spectroscopic analyses revealed that after mutation the far UV CD spectrum of prochymosin was considerably modified, showing less negative ellipticity values, and the fluorescence emission intensities of prochymosin and pseudochymosin were remarkably reduced. The stabilities of prochymosin and pseudochymosin, especially, were dramatically decreased. The stabilization energy of prochymosin was reduced by 7-8 kJ/mol. The inactivation temperature of pseudochymosin was decreased by 15-20 ℃. The wild-type pseudochymosin was stable at pH 1.5 and 6.5, whereas the mutants were completely inactivated at the same pH values. Taken together, it is reasonable to conclude that the 21-24 loop (GTPP) plays an important role in determining the stability of prochymosin and pseudochymosin, although the mutants with mutated loop (GG or SG) still can refold into an active conformation.