Proton transfer from exogenous donors in catalysis by human carbonic anhydrase II.
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 A 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.
Elder
Ileana
I
Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, FL 32610-0267, USA.
Tu
Chingkuang
C
Ming
Li-June
LJ
McKenna
Robert
R
Silverman
David N
DN
D019943:Amino Acid Substitution; D001665:Binding Sites; D002245:Carbon Dioxide / Q000737:chemistry; D024402:Carbonic Anhydrase II / Q000737:chemistry* / Q000235:genetics; D002384:Catalysis; D003035:Cobalt / Q000737:chemistry; D006801:Humans; D007093:Imidazoles / Q000737:chemistry; D010100:Oxygen / Q000737:chemistry; D017354:Point Mutation; D011522:Protons
D007093:Imidazoles; D011522:Protons; D002245:Carbon Dioxide; D003035:Cobalt; D024402:Carbonic Anhydrase II; C023439:4-methylimidazole; D010100:Oxygen