[Affinity labeling of leucine aminopeptidase with new substrate analog inhibitors]

Substrate analogous compounds differing both in type and reactivity (diazonium group, chloro and mo ethyl ketone groups) and in the position of the reactive group at the inhibitor molecule were studied for their adequacy for affinity labelling of leucine amino-peptidase. The chloro methyl ketone derivatives of amino acids with free alpha-amino group are competitive inhibitors. Also, the more reactive bromine compound PheCh2br (Ki 1.2 mM) compared with the PheCH2C1 (Ki 0.3 mM) fails to give an irreversible inactivation of leucine amino-peptidase. The dipeptide derivatives Leu-PheCH2C1 and Phe-LeuCH2C1 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 (pN+/2)CH3 and the two dipeptides Phe(pN+/2)-Phe and Phe(pN+/2)-Phe(pN+/2) with N-terminal diazonium groups afford an irreversible inactivation of leucine aminopeptidase in a time- and concentration-dependent reaction. The Phe-Phe(pN+/2) reactive only C-terminally, is less effective; the inhibition is partly reversible. The inactivation is strongly reduced by the competitive inhibitor Thr(but)-Phe-Pro. These effects are discussed with regard to the specific site of attack of the inhibitors in the active binding centre of leucine aminopeptidase. The synthesis of the three 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.