Kinetics of action of chymosin (rennin) on some peptide bonds of bovine .beta.-casein

Abstract

The first steps of proteolysis of bovine .beta.-casein by chymosin were studied quantitatively by using reverse-phase high-performance liquid chromatography (RP-HPLC). Although chymosin has a broad specificity, it was possible to selectively study the hydrolysis of 2 bonds (Ala-189-Phe-190 and Leu-192-Tyr-193) by choosing appropriate conditions. The disappearance of the substrate and the appearance of the reaction products as a function of time were followed at 220 nm by RP-HPLC. For concentrations where .beta.-casein was in a micellar form, the Michaelian parameters corresponding to the cleavage of bond 192.sbd.193 were determined by measuring initial rates of reaction at different substrate concentrations in a time period for which splitting of bond 189.sbd.190 was negligible. The following results were obtained: .**GRAPHIC**. = 1.54 s-1, .**GRAPHIC**. = 0.075 mM and .**GRAPHIC**. = 20.6 mM-1 s-1. Under conditions where the protein was in a monomeric state, the following parameters were determined for the splitting of bond 192.sbd.193 by integrating the Michaelis equation: .**GRAPHIC**. = 0.056 s-1, .**GRAPHIC**. = 0.007 mM and .**GRAPHIC**. = 79.7 mM-1 s-1. Under the latter conditions the 4 enzymic reactions involved in the cleavage of bonds 189.sbd.190 and 192.sbd.193 were 1st-order reactions. The 4 corresponding apparent rate constants were calculated by using a computer program. Excellent agreement was obtained between concentrations of 4 molecular species measured during the reaction period and those calculated by using the 4 apparent rate constants.