title: Conformational mobility of His-64 in the Thr-200----Ser mutant of human carbonic anhydrase II.
abstract: The three-dimensional structure of the Thr-200----Ser (T200S) mutant of human carbonic anhydrase II (CAII) has been determined by X-ray crystallographic methods at 2.1-A resolution. This particular mutant of CAII exhibits CO2 hydrase activity that is comparable to that of the wild-type enzyme with a 2-fold stabilization of the E.HCO3- complex and esterase activity that is 4-fold greater than that of the wild-type enzyme. The structure of the mutant enzyme reveals no significant local changes accompanying the conservative T200S substitution, but an important nonlocal structural change is evident: the side chain of catalytic residue His-64 rotates away from the active site by 105 degrees about chi 1 and apparently displaces a water molecule. The displaced water molecule is present in the wild-type enzyme; however, the electron density into which this water is built is interpretable as an alternate conformation of His-64 with 10-20% occupancy. The rate constants for proton transfer from the zinc-water ligand to His-64 and from His-64 to bulk solvent are maintained in the T200S variant; therefore, if His-64 is conformationally mobile about chi 1 and/or chi 2 during catalysis, compensatory changes in solvent configuration must sustain efficient proton transfer.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710.
authors.forename: J F
authors.initials: JF
authors.lastname: Krebs
authors.forename: C A
authors.initials: CA
authors.lastname: Fierke
authors.forename: R S
authors.initials: RS
authors.lastname: Alexander
authors.forename: D W
authors.initials: DW
authors.lastname: Christianson
mesh_terms: D002256:Carbonic Anhydrases / Q000737:chemistry*; D003196:Computer Graphics; D004252:DNA Mutational Analysis; D004950:Esterases / Q000737:chemistry; D005583:Fourier Analysis; D006639:Histidine / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D009038:Motion; D011487:Protein Conformation; D012694:Serine / Q000737:chemistry; D013329:Structure-Activity Relationship; D013912:Threonine / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
chemical_list: D008024:Ligands; D013912:Threonine; D012694:Serine; D006639:Histidine; D004950:Esterases; D002256:Carbonic Anhydrases; D015032:Zinc