Neutron Crystallography Reveals How Carbonic Anhydrases (CAs) Work

The Science

Human carbonic anhydrase II (HCA II) catalyzes the reversible hydration of carbon dioxide to form bicarbonate and a proton. CAs are a family of enzymes that play an essential role in the metabolism of carbon dioxide by converting it into a carbonate ion and a proton. Because they are very stable and inexpensive, CAs could be used in significant large-scale applications such as carbon sequestration processes and biofuel production. However, little is known about the arrangement of the active site of CAs while they carry out their function, a gap that has impeded design of optimized CAs for these applications. Neutron crystallography experiments at the Los Alamos Neutron Science Center to determine the structure of human carbonic anhydrase II have revealed the orientation of amino acids around the zinc ion in the active site, as well as the unexpected presence of a water molecule bound to the metal ion. The details of the ordered water structure that occupies the active site are also visible, and their orientations suggest that cleavage of H-bonds and flipping of water molecules are required for the excess proton to leave the active site during catalysis. This is the first report of the neutron structure of any carbonic anhydrase, and the data elucidate interesting features of the active site not previously observed with implications for the proton transfer pathway during catalysis. The zinc-bound solvent appears to be D2O and is involved in two hydrogen bonds to DW and W1. The proton shuttle His64 is oriented predominantly in the inward conformation and is neutral, with the unprotonated NE2 atom pointing toward the zinc-bound D2O, ready to pick up a proton. This structural information has enabled development of a mechanism to explain the proton transfer process and is being used to re-engineer the enzyme to be pH insensitive and thermally stable for carbon sequestration or biodiesel production.

Principal Investigator

S. Zoë Fisher
Los Alamos National Laboratory


Fisher, S. Z., A. Y. Kovalevsky, J. F. Domsic, M. Mustyakimov, R. McKenna, D. N. Silverman, and P. A. Langan. 2010. “Neutron Structure of Human Carbonic Anhydrase II: Implications for Proton Transfer,” Biochemistry 49, 415–21.