J. Biol. Chem., Vol. 263, Issue 20, 9785-9789, 07, 1988

Structure and mechanism of formation of recombinant-derived chymosin C

DE Danley and KF Geoghegan
Pfizer Central Research, Groton, Connecticut 06340.

Chymosin C, an autolysis product of chymosin A, is not formed from chymosin B (Foltmann, B. (1966) C. R. Trav. Lab. Carlsberg 35, 143-231) even though chymosins A and B differ in only a single residue (residue 286 is Asp in chymosin A but is Gly in chymosin B). Autolysis of recombinant-derived chymosin A yielded chymosin C for structural analysis. N-terminal sequencing revealed two peptide chains in chymosin C, one of which begins with Gly43, the N terminus of chymosin A; the other begins with Asp289. C-terminal sequencing, peptide mapping, and amino acid analysis showed that chymosin A undergoes autolytic excision of Asp286--Glu287--Phe288 in yielding chymosin C. As predicted from the established disulfide pattern and verified by size-exclusion chromatography under denaturing conditions, no disulfide bond cross- links the two chymosin A fragments which comprise chymosin C. The N- terminal fragment (243 residues) is termed the C-protein, and the C- terminal fragment (77 residues) is termed the C-peptide. Studies with synthetic peptide analogues of relevant regions of the chymosin sequences suggested that Tyr285--Asp286 is the first chymosin A peptide bond hydrolyzed during chymosin C formation and that Tyr285--Gly286 in chymosin B is not cleaved. Cleavage of Phe288--Asp289, a bond which is present in both chymosins A and B, most likely occurs in the A form as a secondary event following nicking at Tyr285--Asp286. This explains why chymosin A gives rise to chymosin C, but chymosin B does not. Examination of the sequence of a prochymosin gene (Hidaka, M., Sasaki, K., Uozumi, T., and Beppu, T. (1986) Gene (Amst.) 43, 197-203) showed that the autolysis-sensitive region of the polypeptide is encoded by genomic DNA located at the end of one exon and at the beginning of the next. Thus, chymosin C illustrates the correlation of protease- sensitive regions of protein sequences with genomic splice junctions.
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