KINETIC AND STRUCTURAL CHARACTERIZATION OF SPINACH CARBONIC-ANHYDRASE

We have carried out kinetics studies of spinach carbonic anhydrase (CA ) using stopped-flow spectrophotometry at steady state and C-13-NMR ex change at chemical equilibrium. We found that the rate of CO2 reversib le arrow HCO3- exchange catalyzed by spinach CA at pH 7.0 to be 3-5 ti mes faster than the maximal k(cat) for either CO2 hydration or HCO3- d ehydration at steady state, suggesting a rate-determining H+ transfer step in the catalytic mechanism. Correspondingly, we measured a pH-ind ependent solvent deuterium isotope effect on k(cat) of approximately 2 .0, and found that the rate of catalysis was significantly decreased a t external buffer concentrations below 5 mM. Our results are consisten t with a zinc-hydroxide mechanism of action with for spinach CA, simil ar to that of animal carbonic anhydrases. We have also collected X-ray absorption spectra of spinach CA. Analysis of the extended fine struc ture (EXAFS) suggests that the coordination sphere of Zn in spinach CA must have one or more sulfur ligands, in contrast to animal CAs which have only nitrogen and oxygen ligands. The models which best fit the data have average Zn-N(O) distances of 1.99-2.06 Angstrom, average Zn- S distances of 2.31-2.32 Angstrom, and a total coordination number of 4-6. We conclude that animal and spinach CAs are convergently evolved enzymes which are structurally quite different, but functionally equiv alent.