Infrared studies on the mechanism of action of carbonic anhydrase.

By accurately measuring the difference infrared spectrum of Cot-equilibrated bovine carbonic anhydrase against the ethoxzolamideor azide-inhibited enzyme, we have been able to obtain the absorption maximum at 2341 cm-l that is due to the asymmetric stretching of the CO2 molecule loosely bound to a hydrophobic surface at the active site of the enzyme. COz dissolved in water absorbs at 2343.5 cm-l. The observed small shift for bound CO2 can be attributed to a solvent effect. The absence of a larger spectral shift shows that this substrate is not appreciably distorted upon binding by the enzyme. The binding constant of carbonic anhydrase for CO2 may be estimated directly from the difference spectra taken at various CO 2 pressures. Nitrous oxide is found to compete with COz for binding to this enzyme with approximately the same aGnity. In addition to ethoxzolamide, we found that nitrate, bicarbonate, azide, and the other sulfonamide inhibitors also prevent the formation of the enzyme-substrate complex. Azide ion, both free and bound to metal-ion complexes, also absorbs strongly in this infrared range. A comparison between the infrared spectra of azide bound to the diethylenetriamine Zn(II) and Co@) complexes or to the cobalt-enzyme and the corresponding spectrum for the native enzyme shows that the azide ion is coordinate to the Zn(I1) atom of the native enzyme. Examination of the difference spectra in the presence of both azide and CO2 shows that the bound azide sterically interferes with the binding of COz in the hydrophobic cavity adjacent to the Zn(II). Infrared data on the competition of bicarbonate and hydroxide with azide for Zn(II), when combined with the results on COz binding, lead to the formulation of a detailed but extremely simple molecular mechanism for the catalytic action of carbonic anhydrase, i.e. attack of the bound CO2 by the OHgroup coordinated to the zinc ion.