We have carried out kinetics studies of spinach carbonic anhydrase (CA) using stopped-flow spectrophotometry at steady state and 13C-NMR exchange at chemical equilibrium. We found that the rate of CO2<-->HCO3- exchange catalyzed by spinach CA at pH 7.0 to be 3-5 times faster than the maximal kcat for either CO2 hydration or HCO3- dehydration at steady state, suggesting a rate-determining H+ transfer step in the catalytic mechanism. Correspondingly, we measured a pH-independent solvent deuterium isotope effect on kcat of approximately 2.0, and found that the rate of catalysis was significantly decreased at external buffer concentrations below 5 mM. Our results are consistent with a zinc-hydroxide mechanism of action with for spinach CA, similar to that of animal carbonic anhydrases. We have also collected X-ray absorption spectra of spinach CA. Analysis of the extended fine structure (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 A, average Zn-S distances of 2.31--2.32 A, 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 equivalent.

A novel method to synthesize isoquinolones via oxidative annulation of N‐alkoxy benzamides and alkynes using binaphthyl‐stabilized palladium nanoparticles (Pd‐BNP) as catalyst has been developed. This methodology affords various isoquinolone derivatives in good to excellent yields with high regioselectivities in the presence of air as oxidant. N‐Methoxybenzothioamide was also found to undergo oxidative annulation with alkyne successfully and provided a sulfur analogue of isoquinolones in moderate yields. The Pd‐BNP catalyst was easily recovered and reused up to four times without any apparent agglomeration.magnified image

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