Nuclear Magnetic Resonance Studies of Bovine Carbonic Anhydrase. Binding of Sulfonamides to the Zinc Enzyme

Nuclear magnetic resonance line broadening of sulfonamide inhibitors upon their binding to the zinc bovine carbonic anhydrase has been studied at various temperatures and two frequencies. Competition of different inhibitors for the same site was demonstrated. Comparison of the binding constants ratio determined by nuclear magnetic resonance technique with that determined by enzymatic activity confirmed that the broadening effect was due to specific binding to the active site. The dissociation rate constant of the enzyme-inhibitor complexes was found to be greater than 200 sec^-1. Frequency dependence of the broadening indicated a dipolar relaxation mechanism rather than a chemical shift dependent mechanism. The rotational motion of the aromatic rings of several sulfonamide inhibitors was found to be close to that of the whole protein molecule, while that of methyl groups is much faster. Furthermore, it was concluded that, for the methyl protons, the main contribution to the dipolar relaxation is the intramolecular proton-proton interaction, and for the aromatic protons, the interaction with closely adjacent protein protons. The significance of these results to our understanding of the mode of binding is discussed.