Functional implications of the 21-24 loop in recombinant prochymosin

Area of Science:

• Biochemistry
• Protein Engineering
• Enzymology

Background:

• Prochymosin undergoes autocatalytic activation to chymosin, a key enzyme in milk coagulation.
• The 21-24 loop of prochymosin is implicated in its structural integrity and activation process.

Purpose of the Study:

• To elucidate the role of the prochymosin 21-24 amino acid loop (GTPP) in protein stability and activation.
• To investigate the impact of loop mutations on prochymosin's refolding and conversion to pseudochymosin and chymosin.

Main Methods:

• Site-directed mutagenesis was used to replace the GTPP loop residues (21-24) with GG or SG.
• Mutant proteins were expressed in Escherichia coli and refolded.
• Autocatalytic activation studies at pH 2 and pH 5.5 were performed.
• Spectroscopic analyses (CD, fluorescence) and stability assays (temperature, pH) were conducted.

Main Results:

• Mutants, except GTPP(21-24)GS, were expressed and refolded into active prochymosin analogs.
• Mutant pseudochymosins could be further converted to chymosin analogs.
• Mutations significantly reduced protein stability, decreasing stabilization energy and inactivation temperature.
• Mutant pseudochymosins showed complete inactivation at pH 1.5 and 6.5, unlike wild-type.

Conclusions:

• The 21-24 loop (GTPP) is essential for maintaining the stability of both prochymosin and pseudochymosin.
• Despite reduced stability, mutated prochymosin analogs retain the ability to refold into active conformations and undergo activation.
• This suggests a critical role for the loop in stabilizing the active enzyme forms.