Utilization of copper as a paramagnetic probe for the binuclear metal center of phosphotriesterase.
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Bacterial phosphotriesterase catalyzes the hydrolysis of organophosphate triesters. To be active, the enzyme requires that two divalent cations are bound. These metal ions are bound in close proximity to one another as a binuclear center. To characterize the structure and function of the binuclear metal binding sites, we have prepared the copper-substituted enzyme. The kinetic data indicate that this enzyme is essentially inactive toward the hydrolysis of phosphotriesters. The EPR signal arising from the copper-substituted enzyme is nearly axial, with g parallel = 2.24 and g perpendicular = 2.05 and shows at least seven superhyperfine transitions in the g perpendicular region with A perpendicular = 1.45 x 10(-3) cm-1. These splittings are consistent with the direct ligation of more than one nitrogen to the metal center. The average spin quantitation of copper-substituted enzymes are 0.6 spin/Cu, approximately half of that observed for noninteracting Cu2+ ions. The spin intensity increases to ca. 1 spin/Cu when samples are denatured with acid. The binding of metal ions to the designated alpha and beta sites is highly synergistic (i.e., the metal ions bind in pairs). Mixed metal complexes of the type Cu/X and X/Cu were prepared. When X is a diamagnetic ion (Zn2+ or Cd2+), the spin quantitation increases, but when X is the paramagnetic Co2+ ion, the spin quantitation decreases. This behavior indicates that the low spin intensities observed for copper-substituted phosphotriesterase arise from spin-coupling of the two adjacent Cu2+ ions. The addition of dithiothreitol, ascorbate, or dithionite to the copper-substituted phosphotriesterase results in nearly the complete loss of spin intensity. This indicates that the bound coppers can be reduced to the cuprous state.
author list (cited authors)
Chae, M. Y., Omburo, G. A., Lindahl, P. A., & Raushel, F. M.
complete list of authors
Chae, MY||Omburo, GA||Lindahl, PA||Raushel, FM