Inactivation of Pyroglutamyl Aminopeptidase by Na-Carbobenzoxy-L-Pyroglutamyl Chloromethyl Ketone

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Abstract
Pyroglutamyl aminopeptidase [pyrrolidone-carboxylate peptidase: EC 3.4.11.8] from Bacillus amyloliquefaciens was inactivated rapidly and irreversibly by Nα-carboben-zoxy-L-pyroglutamyl chloromethyl ketone (Z-PGCK). The second-order rate constant of the inactivation was 1.1× 105 M−1.S−1, a value which is comparable to that of the clostripain-TLCK reaction. The D-isomer of this chloromethyl ketone derivative was almost inert toward the enzyme under the same conditions. The inactivation reaction was prevented by the presence of a poor substrate, pyroglutamyl-valine. The PCMB-inactivated enzyme, that was reversibly reactivated by 2-mercapto-ethanol, failed to react with Z-PGCK. These results suggest that this chloromethyl ketone derivative reacts as an affinity label, presumably with the active site cysteinyl residue of the enzyme, as was reported for L-pyroglutamyl chloromethyl ketone.