The pH dependency of bovine spleen cathepsin B-catalyzed transfer of N alpha-benzyloxycarbonyl-L-lysine from p-nitrophenol to water and dipeptide nucleophiles. Comparisons with papain.

Cathepsin B has been shown to catalyze the transfer of the N alpha-benzyloxycarbonyl-L-lysyl residue from the corresponding p-nitrophenyl ester substrate to water and dipeptide nucleophiles. These reactions occurred through the formation of an acyl-enzyme intermediate. The pH dependency of the acylation and deacylation steps were determined from the increases in the maximum rate of appearance of p-nitrophenol on addition of glycylglycine or L-leucylglycine to the reaction. The second order acylation rate constant, kcat/Km was found to depend on the state of ionization of three groups in the enzyme having pKa values of 4.2, 5.5, and 8.6. Protonation of the group with pKa = 5.5 decreased but did not abolish enzymatic activity, resulting in the appearance of a second, active protonic form of the enzyme between pH 4.2 and pH 5.5. The first order rate constant for the hydrolysis of the acyl-enzyme intermediate was independent of pH between 4.0 and 7.5. In contrast, acyl group transfer from cathepsin B to glycylglycine and L-leucylglycine depended on a group with a pKa of about 4.5. These results are discussed in terms of possible structural and functional homologies between the active sites of cathepsin B and papain.