title: Proton transfer within the active-site cavity of carbonic anhydrase III.
abstract: The maximal turnover rate of CO2 hydration catalyzed by the carbonic anhydrases is limited by proton transfer steps from the zinc-bound water to solution, steps that regenerate the catalytically active zinc-bound hydroxide. Catalysis of CO2 hydration by wild-type human carbonic anhydrase III (HCA III) (k(cat) = 2 ms (-1)) is the least efficient among the carbonic anhydrases in its class, in part because it lacks an efficient proton shuttle residue. We have used site-directed mutagenesis to test positions within the active-site cavity of HCA III for their ability to carry out proton transfer by replacing various residues with histidine. Catalysis by wild-type HCA III and these six variants was determined from the initial velocity of hydration of CO2 measured by stopped-flow spectrophotometry and from the exchange of 18O between CO2 and H2O at chemical equilibrium by mass spectrometry. The results show that histidine at three positions (Lys64His, Arg67His and Phe131His) have the capacity to transfer protons during catalysis, enhancing maximal velocity of CO2 hydration and 18O exchange from 4- to 15-fold compared with wild-type HCA III. Histidine residues at the other three positions (Trp5His, Tyr7His, Phe20His) showed no firm evidence for proton transfer. These results are discussed in terms of the stereochemistry of the active-site cavity and possible proton transfer pathways.
authors.affiliation: Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL 32610, USA.
authors.forename: Haiqian
authors.initials: H
authors.lastname: An
authors.forename: Chingkuang
authors.initials: C
authors.lastname: Tu
authors.forename: Ke
authors.initials: K
authors.lastname: Ren
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Laipis
authors.forename: David N
authors.initials: DN
authors.lastname: Silverman
mesh_terms: D001665:Binding Sites; D024403:Carbonic Anhydrase III / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D011522:Protons; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D011522:Protons; D011993:Recombinant Fusion Proteins; D024403:Carbonic Anhydrase III