Kinetic and spectroscopic characterization of the gamma-carbonic anhydrasefrom the methanoarchaeon Methanosarcina thermophila

The zinc and cobalt forms of the prototypic gamma-carbonic anhydrase from M ethanosarcina thermophila were characterized by extended X-ray absorption f ine structure (EXAFS) and the kinetics were investigated using steady-state spectrophotometric and (18)0 exchange equilibrium assays. EXAFS results in dicate that cobalt isomorphously replaces zinc and that the metals coordina te three histidines and two or three water molecules. The efficiency of eit her Zn-Cam or Go-Cam for CO2 hydration (k(cat)/K-m) was severalfold greater than HCO3- dehydration at physiological pH values, a result consistent wit h the proposed physiological function for Cam during growth on acetate. For both Zn- and Go-Cam, he steady-state parameter k(cat) for CO2 hydration wa s pH-dependent with a pk(a) of 6.5-6.8, whereas k(cat)/ K-m was dependent o n two ionizations with pk values of 6.7-6.9 and 8.2-8.4. The O-18 exchange assay also identified two ionizable groups in the pH profile of k(cat)/K-m with apparent pK, values of 6.0 and 8.1. The steady-state parameter k(cat) (CO2 hydration) is buffer-dependent in a saturable manner at pH 8.2, and th e kinetic analysis suggested a ping-pong mechanism in which buffer is the s econd substrate. The calculated rate constant for intermolecular proton tra nsfer is 3 x 10(7) M-1 s(-1). At saturating buffer concentrations and pH 8. 5, k(cat) is 2.6-fold higher in H2O than in D2O, suggesting that an intramo lecular proton transfer step is at least partially rate-determining. At hig h pH (pH > 8), k(cat)/K, is not dependent on buffer and no solvent hydrogen isotope effect was observed, consistent with a zinc hydroxide mechanism. T herefore, at high pH the catalytic mechanism of Cam appears to resemble tha t of human CAII, despite significant structural differences in the active s ites of these two unrelated enzymes.