A mutated bovine prochymosin zymogen can be activated without proteolytic processing at low pH.

As a first step towards understanding how the zymogen structure of prochymosin contributes to the process by which active enzyme is produced, we altered the nucleotide sequence which encodes the amino-terminal (or propeptide) region of the protein. Of the two sites for autoproteolysis of prochymosin, one where pseudochymosin is formed at a pH of 2 and the other where chymosin is formed at pH 4-5, we changed the former by removing one codon and changing two other codons. This genetically modified prochymosin was proteolytically processed and activated normally at pH 4.5. However, at pH 2.0 we observed only partial activation of the zymogen and found no evidence of proteolytic processing. The properties of this engineered prochymosin suggest that zymogen activation does not require proteolysis and that the two different zymogen processing sites can function independently from one another.