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. Taste-Active Dipeptides from Hydrolyzed Mushroom Protein Enhance Saltiness
Andrew Moore, Curtis R Luckett, John P Munafo Jr J Agric Food Chem. 2021 Oct 13;69(40):11947-11959. doi: 10.1021/acs.jafc.1c04479. Epub 2021 Oct 4.
An activity-guided fractionation approach applied to thermally treated, enzymatically hydrolyzed mushroom, Agaricus bisporus L., protein led to the identification of several saltiness- and kokumi-enhancing peptides. The identification was accomplished by employing a combination of solid-phase extraction (SPE), gel-permeation chromatography (GPC), and semipreparative reverse-phase high-performance liquid chromatography (RP-HPLC), coupled with sensory analysis. As a result, this study led to the identification of a collection of common mushroom derived tastants, including 5'-mononucleotides and free amino acids, along with several taste-modulating pyroglutamyl dipeptides, including pyroglutamylcysteine (pGlu-Cys), pyroglutamylvaline (pGlu-Val), pyroglutamylaspartic acid (pGlu-Asp), pyroglutamylglutamic acid (pGlu-Glu), and pyroglutamylproline (pGlu-Pro). The taste-modulating thresholds for the pyroglutamyl dipeptides were calculated in a model mushroom broth containing natural concentrations of guanosine 5'-monophosphate and 14 amino acids, all with dose-over-threshold (DoT) factors ≥1. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed to quantitate the pyroglutamyl dipeptides, and their concentrations ranged from 2 to 58 μmol/L; however, they were determined to be present in the hydrolysate below their individual taste-modulating thresholds. Despite being present below their individual thresholds, when the dipeptides were collectively added to a model mushroom broth at their natural concentrations (143 μmol/L combined), both salty (p = 0.0061) and kokumi (p = 0.0025) taste attributes were significantly enhanced, demonstrating a synergistic subthreshold taste-modulating effect. This study lays the groundwork for future investigations on the saltiness-enhancing potential of mixtures of subthreshold levels of pyroglutamyl dipeptides found in mushrooms and other sources.
3. Inactivation of pyroglutamyl aminopeptidase by L-pyroglutamyl chloromethyl ketone
K Fujiwara, T Kitagawa, D Tsuru Biochim Biophys Acta. 1981 Mar 13;658(1):10-6. doi: 10.1016/0005-2744(81)90244-8.
A chloromethyl ketone derivative of pyroglutamic acid was newly synthesized and its reactivity with bacterial pyroglutamyl aminopeptidase (L-pyroglutamyl-peptide hydrolas, EC 3.4.11.8) as an affinity labelling reagent was examined. The compound was found to inactivate the enzyme markedly and rapidly at very low concentrations, though the enzyme was resistant to N-tosyl-phenylalanyl chloromethyl ketone. The rate of the enzyme inactivation by pyroglutamyl chloromethyl ketone was retarded in the presence of a poor substrate, pyroglutamyl valine. The enzyme inactivated by treating with p-chloromercuribenzoate failed to react with pyroglutamyl chloromethyl ketone. These results strongly suggest an active site-directed mechanism for the enzyme inactivation by pyroglutamyl chloromethyl ketone. This compound was shown to be useful as a titrant for the catalytically active protein of pyroglutamyl aminopeptidase.
