Affinity labeling of the active site of aspergillopeptidase B

Aspergillopeptidase B exhibits a strong preference for both the phenyl group and the carbobenzoxy moiety of carbobenzoxyphenylalanine methylester. The chloromethylketones of N⁶-tosyllysine and tosylphenylalanine do not inactivate the enzyme due to low binding affinities. Aspergillopeptidase B is slowly inactivated by the bromomethylketone of carbobenzoxyphenylalanine with an apparent second-order rate constant of 0.16 m^?1 sec^?1 at pH 7.0. A direct correspondence exists between the loss of activity, incorporation of tritiated carbobenzoxyphenylalanine, the disappearance of a histidine residue, and the appearance of a residue of N^r-carboxymethylhistidine. The rate of alkylation by carbobenzoxyphenylalanine is retarded by the competitive inhibitor N²-benzoylarginine.