Investigation of Methionine Sulfoxide Formation as a Regulator of Proteolysis

Abstract

The oxidation status of the methionine in the Tyr-Met cleavage site of von Willebrand Factor affects its rate of proteolysis and therefore oxidation has been proposed as a potential regulator of coagulation. More generally, little is known of the effects of methionine sulfoxide formation at the cleavage site in substrates on protease binding and kinetics. Bovine kappa-casein, a significant milk protein, is cleaved by chymosin between a phenylalanine and a methionine, the latter of which can be oxidized to form a methionine sulfoxide. The methionine is not conserved in camel kappa-casein and both bovine and camel chymosins are produced commercially. This makes it a very interesting model system to examine the potential for the evolution of methionine sulfoxide as a regulatory mechanism controlling protein cleavage. This cleavage reaction is also of considerable importance in its own right both in the nutrition of newborn mammals and in commerce, specifically for its application in cheese making. Peptide substrates of various lengths, matching the sequences of camel and both oxidized and reduced bovine kappa-casein, were digested with bovine and camel chymosin to determine the effects of oxidation upon the enzymatic activities in this system. In addition a comparison was made between the effects of peptide substrate oxidation and whole protein oxidation. Oxidation of substrates caused experimentally significant changes in both Km and kcat, although the effect in the latter was more substantial. Since large effects are observable even in systems not showing conservation of methionine in critical binding sites, this indicates that methionine oxidation could easily become a selection factor, leading to evolution of methionine oxidation as a regulatory mechanism for protein or peptide cleavage.