title: Examination of the role of Gln-158 in the mechanism of CO(2) hydration catalyzed by beta-carbonic anhydrase from Arabidopsis thaliana.
abstract: We have cloned and overexpressed a variant of Arabidopsis thaliana beta-carbonic anhydrase (Q158A) that deletes the functional equivalent of the backbone amide NH of Thr-199 in human alpha-carbonic anhydrase II. The latter residue is hypothesized to be important in catalyzing the rate of CO(2)(-) HCO (3)(-) interconversion in alpha-carbonic anhydrase but this hypothesis is not directly testable in that enzyme. Kinetic studies of a variant of the functionally equivalent residue in A. thaliana beta-carbonic anhydrase provide direct evidence for the role of this residue in beta-carbonic anhydrase. Namely, the mutation of Gln-158 to Ala results in a significant decrease in the maximal k(cat) (33% of wild type) at steady state and the maximal rate of CO(2)(-) HCO(2)(-) exchange at chemical equilibrium as measured by R(1)/[E] (7% of wild type), while leaving the maximal rate of H(+) transfer, as measured by k(cat) at steady state, or R(H(2)O)) at chemical equilibrium, largely unaffected.
authors.affiliation: Department of Chemistry, Colgate University, 13 Oak Drive, Hamilton, NY 13346, USA. RRowlett@mail.colgate.edu
authors.forename: Roger S
authors.initials: RS
authors.lastname: Rowlett
authors.forename: Chingkuang
authors.initials: C
authors.lastname: Tu
authors.forename: Paul S
authors.initials: PS
authors.lastname: Murray
authors.forename: Joy E
authors.initials: JE
authors.lastname: Chamberlin
mesh_terms: D017360:Arabidopsis / Q000201:enzymology*; D002245:Carbon Dioxide / Q000378:metabolism*; D002256:Carbonic Anhydrases / Q000235:genetics / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation; D010100:Oxygen / Q000378:metabolism; D010103:Oxygen Isotopes; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
chemical_list: D010103:Oxygen Isotopes; D005973:Glutamine; D002245:Carbon Dioxide; D002256:Carbonic Anhydrases; D010100:Oxygen