Abstract
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 (k(cat)/K(mdf)) 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 k(cat) for CO2 hydration was pH-dependent with a pK(a) of 6.5-6.8, whereas k(cat)/K(m) 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 k(cat)/K(m) with apparent pK(a) 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 the kinetic analysis suggested a ping-pong mechanism in which buffer is the second substrate. The calculated rate constant for intermolecular proton transfer is 3 x 107 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 intramolecular proton transfer step is at least partially rate-determining. At high pH (pH > 8), k(cat)/K(m) is not dependent on buffer and no solvent hydrogen isotope effect was observed, consistent with a zinc hydroxide mechanism. Therefore, at high pH the catalytic mechanism of Cam appears to resemble that of human CAII, despite significant structural differences in the active sites of these two unrelated enzymes.
Original language | English (US) |
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Pages (from-to) | 13119-13128 |
Number of pages | 10 |
Journal | Biochemistry |
Volume | 38 |
Issue number | 40 |
DOIs | |
State | Published - Oct 5 1999 |
All Science Journal Classification (ASJC) codes
- Biochemistry