title: Human carbonic anhydrase III: structural and kinetic study of catalysis and proton transfer.
abstract: The residue phenylalanine 198 (Phe 198) is a prominent cause of the lower activity of human carbonic anhydrase III (HCA III) compared with HCA II and other isozymes which have leucine at this site. We report the crystal structures of HCA III and the site-directed mutant F198L HCA III, both at 2.1 A resolution, and the enhancement of catalytic activity by exogenous proton donors containing imidazole rings. Both enzymes had a hexahistidine extension at the carboxy-terminal end, used to aid in purification, that was ordered in the crystal structures bound in the active site cavity of an adjacent symmetry-related enzyme. This observation allowed us to comment on a number of possible binding sites for imidazole and derivatives as exogenous proton donors/acceptors in catalysis by HCA III. Kinetic and structural evidence indicates that the phenyl side chain of Phe 198 in HCA III, about 5 A from the zinc, is a steric constriction in the active site, may cause altered interactions at the zinc-bound solvent, and is a binding site for the activation of catalysis by histidylhistidine. This suggests that sites of activation of the proton-transfer pathway in carbonic anhydrase are closer to the zinc than considered in previous studies.
authors.affiliation: Departments of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, Florida 32610, USA.
authors.forename: David M
authors.initials: DM
authors.lastname: Duda
authors.forename: Chingkuang
authors.initials: C
authors.lastname: Tu
authors.forename: S Zoë
authors.initials: SZ
authors.lastname: Fisher
authors.forename: Haiqain
authors.initials: H
authors.lastname: An
authors.forename: Craig
authors.initials: C
authors.lastname: Yoshioka
authors.forename: Lakshmanan
authors.initials: L
authors.lastname: Govindasamy
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Laipis
authors.forename: Mavis
authors.initials: M
authors.lastname: Agbandje-McKenna
authors.forename: David N
authors.initials: DN
authors.lastname: Silverman
authors.forename: Robert
authors.initials: R
authors.lastname: McKenna
mesh_terms: D024403:Carbonic Anhydrase III / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D006639:Histidine / Q000378:metabolism; D006801:Humans; D007093:Imidazoles / Q000737:chemistry; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D011522:Protons; D013329:Structure-Activity Relationship; D013912:Threonine / Q000378:metabolism; D015032:Zinc / Q000737:chemistry
chemical_list: C471213:His-His-His-His-His-His; D007093:Imidazoles; D009842:Oligopeptides; D011522:Protons; D013912:Threonine; D006639:Histidine; C029899:imidazole; D024403:Carbonic Anhydrase III; D007930:Leucine; D015032:Zinc; D008239:Lysine