Characterization of the inhibitor complexes of cobalt carboxypeptidase A by electron paramagnetic resonance spectroscopy.

The metal coordination sphere of cobalt-substituted carboxypeptidase A and its complexes with inhibitors has been characterized by X-band electron paramagnetic resonance (EPR) spectroscopy. The temperature dependence of the EPR spectrum of cobalt carboxypeptidase and the g anisotropy are consistent with a distorted tetrahedral geometry for the cobalt ion. Complexes with L-phenylalanine, a competitive inhibitor of peptide hydrolysis, as well as other hydrophobic L-amino acids all exhibit very similar EPR spectra described by three g values that differ only slightly from that of the cobalt enzyme alone. In contrast, the EPR spectra observed for the cobalt enzyme complexes with 2-(mercaptoacetyl)-D-Phe, L-benzylsuccinate, and L-beta-phenyllactate all indicate an approximately axial symmetry of the cobalt atom in a moderately distorted tetrahedral metal environment. Phenylacetate, beta-phenylpropionate, and indole-3-acetate, which exhibit mixed modes of inhibition, yield EPR spectra indicative of multiple binding modes. The EPR spectrum of the putative 2:1 inhibitor to enzyme complex is more perturbed than that of the 1:1 complex. For beta-phenylpropionate, partially resolved hyperfine coupling (122 x 10(-4) cm-1) is observed on the g = 5.99 resonance, possibly indicating a stronger metal interaction for this binding mode. The structural basis for the observed EPR spectral perturbations is discussed with reference to the existing crystallographic kinetic and electronic absorption, nuclear magnetic resonance, and magnetic circular dichroic data.