Structural and kinetic characterization of an archaeal β-class carbonic anhydrase

The beta -class carbonic anhydrase from the archaeon Methanobacterium thermoautotrophicum (Cab) was structurally and kinetically characterized. Analytical ultracentrifugation experiments show that Cab is a tetramer, Circular dichroism studies of Cab and the Spinacia oleracea (spinach) beta -class carbonic anhydrase indicate that the secondary structure of the beta -class enzymes is predominantly alpha -helical, unlike that of the alpha- or gamma -class enzymes. Extended X-ray absorption fine structure results indicate the active zinc site of Cab is coordinated by two sulfur and two O/N ligands, with the possibility that one of the O/N ligands is derived from histidine and the other from water. Both the steady-state parameters k(cat) and k(cat)/K-m for CO2 hydration are pH dependent. The steady-state parameter k(cat) is buffer-dependent in a saturable manner at both pH 8.5 and 6.5, and the analysis suggested a ping-pong mechanism in which buffer is the second substrate. At saturating buffer conditions and pH 8.5, k(cat) is 2.1-fold higher in H2O than in D2O, consistent with an intramolecular proton transfer step being rate contributing. The steady-state parameter k(cat)/K-m is not dependent on buffer, and no solvent hydrogen isotope effect was observed. The results suggest a zinc hydroxide mechanism for Cab. The overall results indicate that prokaryotic beta -class carbonic anhydrases have fundamental characteristics similar to the eukaryotic beta -class enzymes and firmly establish that the alpha-, beta-, and gamma -classes are convergently evolved enzymes that, although structurally distinct, are functionally equivalent.