Kinetic study of the action of bovine chymosin and pepsin A on bovine kappa-casein.
A study was carried out to determine the Michaelian parameters relative to the action of chymosin and pepsin A on bond Phe105-Met106 of bovine kappa0-casein (carbohydrate-free fraction in micellar state). The reaction was performed in citrate buffer, pH 6.2, at 30 degrees C. The reaction mixture was analysed by reverse phase HPLC. Dosages of peptide 106-169 (caseino macropeptide) at different reaction times from recordings of its absorbance at 220 nm gave the initial rates of reaction at each substrate concentration. From these values the following parameters were determined: kcat = 68.5 s-1, Km = 0.048 mM, kcat/Km = 1,413 mM-1 s-1 for chymosin, and kcat = 45 s-1, Km = 0.018 mM, kcat/Km = 2,439 mM-1 s-1 for pepsin A. For chymosin they are similar to those obtained previously in dimethyl glutarate buffer, pH 6.6, at 30 degrees C, using fragment 98-111 of kappa-casein as substrate. It can thus be concluded that neither the micellar state nor the presence of the whole peptide chain of kappa-casein (our conditions) significantly affect the action of chymosin on fragment 98-111, which seems to contain all information that makes bond 105-106 highly sensitive to chymosin. For pepsin A, only the information contained in fragment 103-108 appears to be required.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002364:Caseins / Q000378:metabolism*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D012085:Chymosin / Q000378:metabolism*; D007700:Kinetics; D010434:Pepsin A / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification
D000596:Amino Acids; D002364:Caseins; D010446:Peptide Fragments; D010434:Pepsin A; D012085:Chymosin