Proton transfer from exogenous donors in catalysis by human carbonic
anhydrase II
Arch. Biochem. Biophys. 2005, 437, 106-114.
Ileana Elder,a Chingkuang Tu,a
Li-June Mig,b Robert McKenna,c and David N. Silvermana
aDepartment of Pharmacology and Therapeutics,
University of Florida College of Medicine, Gainesville, FL 32610-0267, USA
bDepartment of Chemistry and Institute for Biomolecular
Science, University of South Florida, 4202 Fowler Avenue, Tampa, FL 33620-5250,
USA
cDepartment of Biochemistry and Molecular Biology,
University of Florida College of Medicine, Gainesville, FL 32610-0267, USA
Received 9 February 2005; revised 9 February 2005. Available
online 17 March 2005
In the site-specific mutant of human carbonic anhydrase in which the proton
shuttle His64 is replaced with alanine, H64A HCA II, catalysis can be activated
in a saturable manner by the proton donor 4-methylimidazole (4-MI). From
1H NMR relaxivities, we found 4-MI bound as a second-shell ligand
of the tetrahedrally coordinated cobalt in Co(II)-substituted H64A HCA II,
with 4-MI located about 4.5 
from the metal. Binding constants of 4-MI to H64A HCA II were estimated
from: (1) NMR relaxation of the protons of 4-MI by Co(II)-H64A HCA II, (2)
the visible absorption spectrum of Co(II)-H64A HCA II in the presence of
4-MI, (3) the inhibition by 4-MI of the catalytic hydration of CO2,
and (4) from the catalyzed exchange of 18O between CO2
and water. These experiments along with previously reported crystallographic
and catalytic data help identify a range of distances at which proton transfer
is efficient in carbonic anhydrase II.
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