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H2CO3 as Substrate for Carbonic Anhydrase in the Dehydration of HCO3 -
Seymour H. Koenig and Rodney D. Brown
Proceedings of the National Academy of Sciences of the United States of America
Vol. 69, No. 9 (Sep., 1972), pp. 2422-2425
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/61783
Page Count: 4
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Carbonic anhydrase, a metalloenzyme containing one zinc atom per protein molecule of molecular weight 30,000, catalyzes the interconversion of CO2 and HCO3 - in solution. The rate of catalysis, among the fastest known, is pH-dependent, with a pKEnz near neutral. Arguments are presented to show that: (i) only the high-pH form of the enzyme is active both for the hydration and dehydration reactions (ii) at high pH there is an H2O ligand on the metal (not an OH- as is often argued), and (iii) the substrate for the dehydration reaction is the neutral H2CO3 molecule. The arguments are based on data in the literature on the nuclear relaxation rates of Cl- ions and water protons in solutions of carbonic anhydrase, on strict application of the principle of microscopic reversibility, and on kinetic considerations. It has been argued that H2CO3 cannot be the substrate for the dehydration reaction because the observed CO2 production rate is somewhat faster than the maximum rate at which H2CO3 molecules can diffuse to the active site of the enzyme. However, current models that consider HCO3 - as the substrate implicitly require that protons diffuse to the enzyme at an even greater rate, well outside the limitations imposed by diffusion. We consider two mechanisms to obviate the diffusion limitation problem, and conjecture that at high substrate concentration, H2CO3 reaches the active site by collision with the enzyme molecule, and subsequent surface diffusion to the active site. At lower substrate concentrations, corresponding to [HCO3 -] <1 mM, generation of H2CO3 molecules near the enzyme by the recombination reaction H+ + HCO3 -→ H2CO3 can supply an adequate flux of substrate to the active site.
Proceedings of the National Academy of Sciences of the United States of America © 1972 National Academy of Sciences