Kinetic and Spectroscopic Characterization of the Gamma-Carbonic Anhydrase from the Methanoarchaeon Methanosarcina thermophila†

The zinc and cobalt forms of the prototypic γ-carbonic anhydrase from Methanosarcina thermophila were characterized by extended X-ray absorption fine structure (EXAFS) and the kinetics were investigated using steady-state spectrophotometric and 18O exchange equilibrium assays. EXAFS results indicate that cobalt isomorphously replaces zinc and that the metals coordinate three histidines and two or three water molecules. The efficiency of either Zn−Cam or Co−Cam for CO2 hydration (kcat/Km) was severalfold greater than HCO3- dehydration at physiological pH values, a result consistent with the proposed physiological function for Cam during growth on acetate. For both Zn− and Co−Cam, the steady-state parameter kcat for CO2 hydration was pH-dependent with a pKa of 6.5−6.8, whereas kcat/Km was dependent on two ionizations with pKa values of 6.7−6.9 and 8.2−8.4. The 18O exchange assay also identified two ionizable groups in the pH profile of kcat/Km with apparent pKa values of 6.0 and 8.1. The steady-state parame...