On the reaction of diazoacetyl compounds with pepsin.

Previous studies have shown that diazoacetyl-DL-norleutine methyl ester inactivates pepsin, that Cu(I1) greatly facilitates the reaction, and that in the presence of Cu(I1) substitution occurs specifically at the carboxyl group of an aspartic acid residue in the enzyme. The present experiments were designed to establish the reasons for the speed and specificity of the reaction and the role of the metal ion. Ag(1) proves to be as effective as Cu(I1) in promoting the reaction, whereas Cd(II), Co(II), Pb(II), and Zn(I1) are not effective. Au(III) inactivates pepsin in the absence of a diazo compound. If Cu(I1) and the diazo compound are mixed prior to the addition of pepsin, the rate of inactivation is increased, suggesting that a reactive complex is formed. The pH optimum for inactivation in the presence of Cu(I1) is pH 5.5 to 5.8, whereas with Ag(1) it is pH 4.9. If the diazo compound and Cu(I1) are mixed prior to the addition of pepsin, the pH optimum is reduced to about pH 5.0. The diazotized inhibitor need not be a substrate analogue, for diazoacetylglycine ethyl ester serves as well as the norleucine derivative. Diazoacetic acid methyl ester also inactivates pepsin rapidly in the presence of Cu(II), but more than 1 eq of reagent is incorporated. Although the rates of inactivation of pepsin prepared from pepsinogen and of commercial pepsin are similar, the stoichiometry of the reaction is not. More diazoacetyl compound is incorporated into commercial pepsin, which is known to be heterogeneous, than into pepsin prepared from pepsinogen. It is suggested that a copper-complexed carbene is the reactive species. This hypothesis is supported by the finding that dimethylsulfonium phenacylide, which is known to form such a complex, rapidly inactivates pepsin in the presence of Cu(I1). Reaction of a carbene is thought to occur at a protonated carboxyl and the specificity is determined by the proximity of an ionized carboxyl which serves to orient the positively charged inhibitor molecule. The proposed mechanism of inactivation is compatible with the presence at or near the active site of pepsin of two carboxyl groups of markedly different pK values. One of these carboxyl groups has a pK higher than either of the two groups which previous kinetic