1. Inactivation of pyroglutamyl aminopeptidase by N alpha-carbobenzoxy-L-pyroglutamyl chloromethyl ketone
K Fujiwara, E Matsumoto, T Kitagawa, D Tsuru J Biochem. 1981 Aug;90(2):433-7. doi: 10.1093/oxfordjournals.jbchem.a133490.
Pyroglutamyl aminopeptidase [pyrrolidone-carboxylate peptidase: EC 3.4.11.8] from Bacillus amyloliquefaciens was inactivated rapidly and irreversibly by N alpha-carbobenzoxy-L-pyroglutamyl chloromethyl ketone (Z-PGCK). The second-order rate constant of the inactivation was 1.1 x 10(5) 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-mercaptoethanol, 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.
2. Production of "internal surface reversed-phase" supports: the hydrolysis of selected substrates from silica using chymotrypsin
I H Hagestam, T C Pinkerton J Chromatogr. 1986 Oct 10;368(1):77-84. doi: 10.1016/s0021-9673(00)91048-3.
The degree of hydrolysis of substrates attached to silica supports with alpha-chymotrypsin has been evaluated relative to the production of "internal surface reversed-phase" supports. The peptide substrates N-tert.-butoxycarbonyl-L-phenylalanine (Boc-L-Phe), N-carbobenzoxy-L-valine-L-phenylalanine, N-acetyl-L-phenylalanine (acetyl-L-Phe) and N-benzoyl-L-phenylalanine as well as phenylpropionic acid were attached to glycerylpropyl-bonded silica via a diamine spacer using 1,1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as a coupling catalyst. The products released from the silica support on enzyme treatment were quantified by high-performance liquid chromatography. Boc-L-Phe and acetyl-L-Phe were successfully cleaved from the rigid silica matrix in high yields, whereas the remaining substrates were hydrolyzed to a lesser extent.
3. N-alpha-carbobenzoxy pyroglutamyl diazomethyl ketone as active-site-directed inhibitor for pyroglutamyl peptidase
K Fujiwara, E Matsumoto, T Kitagawa, D Tsuru Biochim Biophys Acta. 1982 Apr 3;702(2):149-54. doi: 10.1016/0167-4838(82)90496-4.
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.
