Nuclear Magnetic Relaxation Dispersion in Protein Solutions: IV. PROTON RELAXATION AT THE ACTIVE SITE OF CARBONIC ANHYDRASE

Abstract

Abstract Bovine and human B erythrocyte carbonic anhydrases substituted with Co2+ cause an enhancement of the nuclear magnetic relaxation rate of solvent water protons (T1-1) at high pH that is decreased by the addition of carbonic anhydrase inhibitors such as azide and Ethoxzolamide. The part of T1-1 which can be inhibited by Ethoxzolamide is due to exchangeable protons located at the active site of the enzyme. This inhibitable part of T1-1 is pH-dependent with a pK of 7.0 ± 0.2 for the bovine cobalt enzyme and a pK of 8.2 ± 0.2 for the human B cobalt enzyme. From the magnetic field dependence of the inhibitable part of T1-1, a correlation time for the dipolar interaction of a proton with a cobalt electronic spin of 10-11 sec, a proton-cobalt distance of 2.5 to 2.9 A, and for the bovine enzyme a proton residence time, τm, small compared to 10-5 sec are calculated. We conclude from the pH dependence and the proton-cobalt distance that the proton which contributes to the inhibitable part of T1-1 is located either on a hydroxide ion bound to the Co2+ at the active site or on a water molecule with 1 proton hydrogen bonded to a nearby residue. In contrast to the similarity of the inhibitable part of T1-1 for the bovine and human B cobalt enzymes, there is a non-inhibitable, pH-independent contribution resulting from the cobalt substitution, relatively large for the bovine enzyme and very small for the human B enzyme, that is not understood. The contributions of the (diamagnetic) native zinc enzymes to T1-1 have also been measured. As in the case of apotransferrin (Koenig, S. H., and Schillinger, W. E., J. Biol. Chem., 244, 3283 (1969)), the results agree qualitatively but not quantitatively with theory.