Paramagnetic carbon-13 NMR relaxation studies on the kinetics and mechanism of the bicarbonate ion/carbon dioxide exchange catalyzed by manganese(II) human carbonic anhydrase I

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Abstract
A detailed analysis of the stability and activity of Mn(II) human carbonic anhydrase I and the kinetics and mechanism of its catalysis of the HCO3-/CO2 exchange have been performed at pH 8.5. The analysis was based on the paramagnetic relaxation rates R1p and R2p of the 13C atom of HCO3- in the Mn2+/apoenzyme/HCO3-/CO2 system and the HCO3- .fwdarw. CO2 interconversion rate obtained by the magnetization-transfer technique. The R1p and R2p rates were measured as functions of the temperature, magnetic field strength, and substrate and apoenzyme concentrations and were interpreted on the basis of the Solomon-Bloembergen-Morgan theories and general equations for the ligand exchange [Led, J. J., and Grant, D. M.(1977) J. Am. Chem. Soc. 99, 5845-5858]. From the analysis of the data, a formation constant for the Mn(II) enzyme of log .**GRAPHIC**. = 5.8 .+-. 0.4 was obtained while the activity of the Mn(II) enzyme, measured as the HCO3-/CO2 interconversion rate at [HCO3-] = 0.100 M and pH 8.5, was found to be about 4% of that of the native Zn(II) enzyme. However, an effective dissociation constant .**GRAPHIC**. .ltorsim. 12 mM and a maximal exchange rate constant .**GRAPHIC**. .simeq. 400 s-1, also derived by the analysis, result in an apparent second-order rate constant .**GRAPHIC**. only a factor of 4 smaller than the corresponding rate constant for the native Zn(II) isoenzyme I. Most conspicuously, the resulting distance of only 2.71 .+-. 0.03 .ANG. between the Mn2+ ion of the enzyme and the 13C atom of HCO3- in the enzyme-bicarbonate complex indicates that the bicarbonate is bound to the metal ion by two of its oxygen atoms in the central catalytic step, thereby supporting the modified Zn(II)-OH mechanism [Lindskog, S., Engberg, P., Forsman, C., Ibrahim, S. A., Jonsson, B.-H., Simonsson, I., and Tibell, L. (1984) Ann. N.Y. Acad. Sci. 429, 61-75 (and references cited therein)]. In contrast, this binding mode differs from the structure of the complexes suggested in the rapid-equilibrium kinetic model [Pocker, Y., and Deits, T.L. (1983) J. Am. Chem. Soc. 105, 980-986; Pocker, Y., and Deits, T. L. (1984) Ann. N.y. Acad. Sci 429, 76-83].

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