1H and13C NMR studies of the spatial arrangement ofp-nitrophenol and acetate at the catalytic sites of Mn(II)-and Co(II)-substituted human carbonic anhydrase C
Nuclear relaxation studies were performed on Mn2+ and Co2+-substituted human carbonic anhydrase C (HCAC) to determine the active site configurations of the products ofp-nitrophenylacetate hydrolysis. Metal-ligand distances calculated from the magnitudes of the paramagnetic effect on longitudinal nuclear relaxation rates indicate direct coordination of the phenolic oxygen ofp-nitrophenol to the enzyme-bound Mn2+ in the ternary HCAC-Mn2+-p-nitrophenol complex. The Mn2+ top-nitrophenol distances in the quaternary HCAC-Mn2+-acetate-p-nitrophenol and HCAC-Co2+-p-nitrophenol-acetate complexes are also consistent with coordination ofp-nitrophenol to the enzyme-bound metal via the phenolic oxygen. However, the orientation of the aromatic ring ofp-nitrophenol appears to be different for the Co2+ and Mn2+-enzymes. The carboxyl carbon of acetate is in the range of innersphere distances expected for direct coordination of a carboxyl oxygen to the enzyme-bound Mn2+, and the acetate molecule is folded around the metal. Although direct coordination of acetate to the metal in the HCAC-Co2+-p-nitrophenol-acetate complex cannot be inferred from our data, weak innersphere complexation with a somewhat longer Co2+-carboxyl oxygen bond distance is not excluded. We found small but significant differences in the arrangement of the products at the active sites of the Co2+ and Mn2+-enzymes which may be responsible for the differences in their reactivities in the hydrolysis reaction.