Reflections on Edsall's carbonic anhydrase: paradoxes of an ultra fast enzyme.

John Edsall's investigations of human erythrocyte carbonic anhydrase, a zinc metalloenzyme that powerfully catalyzes the reversible hydration of carbon dioxide, highlighted a conundrum regarding the correct hydration product. The measured kinetic parameters could not be reconciled with the choice of carbonic acid, since its bimolecular recombination rate with enzyme would exceed the diffusion limit. The alternate choice of bicarbonate obviated the recombination rate problem but required that the active site deprotonation exceed the diffusion-limited maximum rate by an even greater extent. This paradox was resolved in favor of bicarbonate when the unsuspected role of buffer species indirectly deprotonating the enzyme was finally proposed, spurring numerous investigations to verify the hypothesis. Edsall's laboratory also reported the accidental discovery of the first competitive inhibitor, imidazole. This opened new avenues to understanding the binding of the CO(2) substrate and stimulated many investigations on this inhibitor. Paramagnetic NMR and crystallographic studies demonstrated that the only other known competitive inhibitor, phenol, apparently shared this unusual binding site. Despite enormous progress since Edsall's retirement, particularly the use of site-directed mutagenesis approaches, the precise interactions of carbon dioxide and bicarbonate with specific active site moieties remain as elusive today as when Edsall first considered these questions.

A comparative approach to carbonic anhydrase: the work of Thomas H. Maren.

Thomas H. Maren studied carbonic anhydrase (CA) for half a century, venturing into all aspects of this powerful enzyme from active site chemistry to clinical medicine. He was a keen proponent of comparative physiology to illuminate basic principles of the chemistry and biology of CA and spent 47 summers at the Mt. Desert Island Biological Laboratory (MDIBL) studying many non-mammalian species. Following the venerable strategy of selecting the right creature to explore a particular question, Maren derived important insights into the role of CA in ion transport, acid-base regulation and gas exchange. Using the fact that tissue CAs are expressed variably in different species, and that these animals differ in temperature, acid-base status and metabolic rate, he defined the contributions of un-catalyzed and catalyzed CO(2) reactions in many physiological processes. Often this strategy simplified a problem and offered answers not easily obtainable in mammals. As examples, he verified the primary role of HCO(3)(-) as lead ion in CSF formation in fish and extended this to mammals. Using marine fish whose kidneys have very little CA, he uncovered mechanisms of acid-base transfer independent of CA that help to explain why CA inhibition does not lead to total bicarbonate depletion.