Distribution and Evolution

There are five evolutionary unrelated classes of CAs: a-, b-, g- ,d-, and e-. Our Lab studies the a-class CAs which are found mainly found in mammals but has also been found in mosquitos and some plant algae. These CAs are considered single-domain mixed a/b globular proteins, that are made of a single polypeptide chain (unmodified molecular weight ~ 29 kD) that act as monomers with one Zn²⁺ per molecule. There are 14 genetically distinct a- isoforms in humans (HCA I to XIV) and their percentage sequence identity varies from 21 to 62% when compared to each other. They differ with regards to expression patterns, expression levels, subcellular location, kinetic properties and sensitivity to inhibitors. Our lab studies mainly human isoforms of CA (HCA II, III, VI, IX, and the CA-Related Proteins VIII, X, and XI) but we also study 2 CAs recently isolated from mosquitoes, AaCA1 (from Aedes aegypti) and AgCA4 (from Anopheles gambiae).

General Function

General functions of CAs are diverse and include renal and male reproductive duct acidification, modulation of hemoglobin’s affinity for O₂ in respiration, acid/base balance, gluconeogenesis (supplies HCO₃^- to pyruvate carboxylase for glucose production), ureagenesis (supplies HCO₃^- to carbamoyl phosphate synthetase for urea production), ion transport/regulation (Na⁺/H⁺ and Cl^-/HCO₃^- exchange), gastric acid production and carbon fixing reactions in plants.

Catalysis

CAs have been extensively studied since the discovery of erythrocyte CA II over seventy years ago. CA catalyzes the reversible hydration/dehydration reaction of CO₂. The first step is the binding of CO₂ in the hydrophobic region of the CA active site. Next is a nucleophilic attack by the zinc-bound OH^- on the substrate carbon to form HCO₃^- . The HCO₃^- is then displaced from the active site by H₂O. The second part of the reaction catalyzed by CA is the rate-limiting step and involves the intra- and inter-molecular transport of a proton. The intramolecular proton transport occurs between the ZnH₂O and the side chain of His64 through an intervening chain of hydrogen-bonded water molecules. His64 sits on the edge of the active site and is ~8 Ĺ away from the ZnOH^-/H₂O. In the crystal structures of HCA II at various pHs, it has been observed that this proton shuttle can occupy dual conformations. The two conformations are the so-called “in” and “out” positions and it has been suggested that this flexibility is required for efficient proton transferThe intermolecular transport step regenerates the active form of the enzyme when His64 transfers a proton to the bulk solvent.

Our Focus

Our lab has many projects in conjunction with the Silverman Lab that involves all aspects of CA structure, catalysis, and function. We are interested in using various structural techniques (from X-ray crystallography, neutron diffraction to molecular modeling) to study the detailed structure of wild type and various mutants of CA.

Selected Recent Publications

Budayova-Spano, M., S. Z. Fisher, M.-T. Dauvergne, M. Agbandje-McKenna, D. N. Silverman, D. A. A. Myles, R. McKenna. 2006. Production and X-ray crystallographic analysis of fully deuterated human carbonic anhydrase II. Acta Cryst., F62: 6-9.
Budayova-Spano 2006

Fisher, S. Z., J. A. Hernandez Prada, C. Tu, D. M. Duda, C. Yoshioka, H. An, L. Govindasamy, D. N. Silverman, R. McKenna. 2005. Structural and kinetic characterization of active-site histidine as a proton shuttle in catalysis by human carbonic anhydrase II. Biochemistry, 44:1097-1105.
Fisher 2005

Duda, D.M. C.K. Tu, S.Z. Fisher, H. An, C. Yoshioka, L. Govindasamy, P. J. Laipis, M. Agbandje-McKenna, D. N. Silverman, R. McKenna. 2005. Human carbonic anhydrase III: Structural and kinetic study of catalysis and proton transfer. Biochemistry, 44:10046-10053.
Duda 2005