Inhibition of bovine lens leucine aminopeptidase by bestatin: number of binding sites and slow binding of this inhibitor.

Aminopeptidases catalyze the hydrolysis of amino acid residues from the amino terminus of peptide substrates. Their activity has been implicated in myriad fundamental biochemical and physiological processes, and alterations in aminopeptidase activity have been correlated with a variety of pathologies. Nevertheless, information about this group of proteases is less well developed. Bovine lens leucine aminopeptidase (blLAP) can be considered prototypical of many enzymes in this family of peptidases. It shows common features of (1) requiring divalent metal ions for activity, (2) having a relatively large size, and (3) having slow, relatively tight binding of bestatin, a transition-state analog of the substrate PheLeu. Bovine lens LAP is the only bestatin-inhibitable aminopeptidase for which structural and mechanistic data are available. However, full exploitation of these data required knowledge of the number of inhibitor molecules bound per subunit. Independent direct binding experiments and kinetic determinations indicate that one bestatin is bound per subunit in blLAP. Ki and Ki* for formation of the initial and final complexes are approximately 1.1 x 10(-7) and 1.3 x 10(-9) M, respectively. The mode of binding is slow and competitive. The t1/2 for formation and deformation of the final enzyme-inhibitor complex is approximately 30 and 22 min, respectively, with 10(-8) M bestatin. To perform these measures, a new assay using physiological peptides (LeuGlyGly) as substrate was adapted. Taken together with prior NMR, photoaffinity labeling, and crystallographic data, these binding data allow us to propose a mechanism of the blLAP-catalyzed hydrolysis of peptides.