Activity of copper-substituted carboxypeptidase A toward oligopeptides and depsipeptides.

Abstract

Cu(II)-substituted carboxypeptidase A catalyzes the hydrolysis of oligopeptides and their depsipeptide (ester) analogues. Stopped-flow fluorescence assays demonstrate that relative to the zinc enzyme the Cu enzyme can have kcat/Km values up to 24% toward esters but only up to 2.5% toward the corresponding peptides. Adding Zn(II) to the copper enzyme reveals a slow exchange process that correlates with an increase in peptidase activity and with changes in the Cu(II) electron paramagnetic resonance spectra. Low concentrations of 1,10-phenanthroline (OP) (0.1-2.5 microM) markedly increase activity toward furanacryloyl-Phe-Phe (up to 8% of the zinc enzyme), but higher concentrations inhibit, resulting in complete inhibition at 0.8 mM OP. The non-metal-binding, hydrophobic analogues m- and p-phenanthroline are only activators of peptide hydrolysis, even at 1 mM. Activation is likely due to a modifier binding to a hydrophobic locus and either displacing an inhibitory peptide binding mode or inducing a conformational change in the active site.