N-alpha-carbobenzoxy pyroglutamyl diazomethyl ketone as active-site-directed inhibitor for pyroglutamyl peptidase

Pyroglutamyl-peptidase (L-pyroglutamyl-peptide hydrolase, EC 3.4.19.3) from Bacillus amyloliquefaciens was covalently labeled with a newly synthesized N-carbobenzoxy-L-pyroglutamyl diazomethyl ketone (Z-PGDK) and was completely inactivated. The inactivation reaction proceeded in pseudo-first order. The kinetic studies demonstrated a rate-limiting step in the inhibition reaction, resulting in the formation of a reversible (enzyme.reagent) complex. The calculated KI,app is 0.12 mM at pH 7.58. The rate of inactivation was pH dependent with an extrapolated pK value of approx. 8.6. The enzyme could be protected against inactivation by a poor substrate, pyroglutamyl-valine. The PCMB-inactivated enzyme, that could be reversibly reactivated by mercaptoethanol, failed to react with Z-PGDK. The enzyme was insensitive toward the D-isomer of Z-PGDK and other diazomethyl ketone derivatives of carbobenzoxy amino acids such as Z-L-proline and Z-L-phenylalanine. These results strongly suggest that the Z-PGDK reacts as an affinity label, presumably with a cysteine residue as the site of alkylation in pyroglutamyl-peptidase, as was reported for chloromethyl ketone derivatives of pyroglutamic acid and its N-carbobenzoxy derivative.