[Affinity labeling and differential labeling of leucine aminopeptidase with diazopeptide inhibitors]

Specific modification of amino acid residues in the active center of LAP by the substrate-like diazonium peptide L-Phe(pN2+)-L-Phe was investigated using the technic of affinity and differential labeling. Prelabeling of the enzyme was performed with the diastereomer D-Phe(pN2+)-L-Phe in the presence of the competitive inhibitor L-Thr(But)-L-Phe-L-Phe (Ki 9.4 x 10(-5) mol/l). The amino derivative L-Phe(pNH2)-L-Phe is a substrate of the LAP with Km 1.2 x 10(-4) mol/l and D-Phe(pNH2)-L-Phe a non-competitive inhibitor with Ki 4.0 x 10(-3) mol/l. The decrease of activity by covalent modification of tyrosine and histidine side chains was irreversible and affects only the catalytic step, whereas the substrate binding was unimpaired. The affinity labeling as well as the differential labeling in presence of Thr(But)-Phe-Phe showed that up to 12 potential covalent modification sites of LAP can be protected.