title: Interaction and influence of phenylalanine-198 and threonine-199 on catalysis by human carbonic anhydrase III.
abstract: Site-directed mutants of human carbonic anhydrase III were used to examine the role of Thr-199 and its interaction with Phe-198 in the catalyzed hydration of CO2. Threonine-199 is a hydrogen bond acceptor for the zinc-bound water, and Phe-198 forms part of the hydrophobic side of the active-site cavity of carbonic anhydrase III. Catalytic activity for a total of five single and double mutants at residues 198 and 199 was determined by stopped-flow spectrophotometry and 18O exchange between CO2 and water measured by mass spectrometry. The replacement Thr-199-->Ala resulted in a 4-fold decrease in the kcat/Km for hydration of CO2. We tested the hypothesis that the 25-fold increase in the kcat/Km for hydration of CO2 accompanying the replacement Phe-198-->Leu in isozyme III is caused by changes in the interaction of Thr-199 with the zinc-bound water or the transition state for catalysis. Comparison of hydration of CO2 by the single and double mutants of isozyme III containing the replacements Thr-199-->Ala and Phe-198-->Leu was consistent with an interaction between these two sites.
authors.affiliation: Department of Pharmacology and Therapeutics, University of Florida, Gainesville 32610.
authors.forename: X
authors.initials: X
authors.lastname: Chen
authors.forename: C
authors.initials: C
authors.lastname: Tu
authors.forename: P V
authors.initials: PV
authors.lastname: LoGrasso
authors.forename: P J
authors.initials: PJ
authors.lastname: Laipis
authors.forename: D N
authors.initials: DN
authors.lastname: Silverman
mesh_terms: D000595:Amino Acid Sequence; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010103:Oxygen Isotopes; D010649:Phenylalanine; D013912:Threonine
chemical_list: D010103:Oxygen Isotopes; D013912:Threonine; D010649:Phenylalanine; D002256:Carbonic Anhydrases