The chemical and kinetic consequences of the modification of papain by N-bromosuccinimide

Abstract

Nonactivated papain was treated with N-bromosuccinimide at pH 4.75. The N-bromosuccinimide-modified enzyme was characterized by (1) the change in absorbance at 280 nm, (2) amino acid analysis, (3) separate chemical determinations of tryptophan and tyrosine, (4) difference spectroscopy, and (5) an N-terminal residue determination. It is concluded that N-bromosuccinimide in sevenfold molar excess oxidizes one tryptophan and two to three tyrosine residues per molecule of nonactivated papain, without causing peptide chain cleavage. Kinetic studies with several substrates and competitive peptide inhibitors were performed at pH 6 using the N-bromosuccinimide-modified papain. In addition, the kinetics of the modified enzyme with the substrate α-N-benzoyl-L-arginine ethyl ester were studied in the region of pH 3.5–9.0. All substrates (and inhibitors) tested, with the exception of α-N-benzoyl-L-arginine p-nitroanilide, displayed approximately a twofold decrease in both kcat and Km (or Ki), relative to the native enzyme. It is concluded that the key tryptophan residue which is modified is probably Trp-177.