Histidine-200 alters inhibitor binding in human carbonic anhydrase B. A carbon-13 nuclear magnetic resonance identification.
We have previously prepared 13C-enriched NT-carboxymethylhistidine-200 human carbonic anhydrase B (CmHCAB) by reacting the native enzyme with 90% [1-13C]bromoacetate (Strader, D.J., and Khalifah, R.G. (1976), J. Am. Chem. Soc. 98, 5043). The 13C nuclear magnetic resonance signal of the enriched carboxylate of CmHCAB proved sensitive to active-site events, permitting, among other things, the determination of the microscopic pKa of the modified histidine. This report extends the study to the complexes of CmHCAB with the inhibitors iodide and azide. It is found that the pKa of histidine-200 is significantly increased when these inhibitors bind. A quantiative comparison of the iodide-induced pKa shift with literature data (Whitney, P. L., and Brandt, H. (1976), J. Biol, Chem. 251, 3862) showing that the binding of iodide is influenced by the ionization of an active-site group of pKa 6.1 allowed the clear identification of histidine-200 as the perturbing group. Other important implications of the magnetic resonance results are also discussed.
D000085:Acetates; D001665:Binding Sites; D002256:Carbonic Anhydrases / Q000097:blood; D006639:Histidine; D006801:Humans; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D011485:Protein Binding; D011487:Protein Conformation
D000085:Acetates; D006639:Histidine; D002256:Carbonic Anhydrases