EXPERIMENTS ON AFFINITY LABELING OF LEUCINE AMINOPEPTIDASE WITH NEW SUBSTRATE ANALOGOUS INHIBITORS

  • 1 January 1976
    • journal article
    • research article
    • Vol. 35  (3-4) , 365-378
Abstract
Analogous substrate compounds differing both in type and reactivity (diazonium group, chloro and bromo ethyl ketone groups) and in the position of the reactive group at the inhibitor molecule were studied for their adequacy for affinity labeling of leucine aminopeptidase [EC 3.4.11.1] [bovine eye lense]. The chloro methyl ketone derivatives of amino acids with free .alpha.-amino group are competitive inhibitors. The more reactive Br compound PheCH2Br (Ki [inhibition constant] 1.2 mM) compared with the PheCH2Cl (Ki 0.3 mM) fails to give an irreversible inactivation of leucine aminopeptidase. The dipeptide derivatives Leu-PheCH2Cl and Phe-LeuCH2 also inhibit the enzyme activity up to 65%, but they are split at the peptide bond under reactivation of the enzyme. p-Diazophenylalanine methylketone Phe(pN2+)CH3 and the 2 dipeptides Phe(pN2+)-Phe and Phe(pN2+)-Phe(pN2+) with N-terminal diazonium groups afford an irreversible inactivation of leucine aminopeptidase in a time- and concentration-dependent reaction. The Phe-Phe(pN2+) reactive only C-terminally, is less effective; the inhibition is partly reversible. The inactivation is strongly reduced by the competitive inhibitor Thr(but)[tert butyl]-Phe-Pro. These effects are discussed with regard to the specific site of attack of the inhibitors in the active bindng center of leucine aminopeptidase. The synthesis of the 3 parasubstituted amino derivatives of phenylalanyl-phenylalanine Phe(pNH2)-Phe, Phe-Phe(pNH2) and Phe(pNH2)-(Phe(pNH2) and their selective conversion to the respective diazonium peptides, retaining the aliphatic .alpha.-amino group, are discussed.