title: Role of Trp19 and Tyr200 in catalysis by the γ-class carbonic anhydrase from Methanosarcina thermophila.
abstract: Although widely distributed in Nature, only two γ class carbonic anhydrases are reported besides the founding member (Cam). Although roles for active-site residues important for catalysis have been identified in Cam, second shell residues have not been investigated. Two residues (Trp19 and Tyr200), positioned distant from the catalytic metal, were investigated by structural and kinetic analyses of replacement variants. Steady-state k(cat)/K(m) and k(cat) values decreased 3- to 10-fold for the Trp19 variants whereas the Y200 variants showed up to a 5-fold increase in k(cat). Rate constants for proton transfer decreased up to 10-fold for the Trp19 variants, and an increase of ~2-fold for Y200F. The pK(a) values for the proton donor decreased 1-2 pH units for Trp19 and Y200 variants. The variant structures revealed a loop composed of residues 62-64 that occupies a different conformation than previously reported. The results show that, although Trp19 and Y200 are non-essential, they contribute to an extended active-site structure distant from the catalytic metal that fine tunes catalysis. Trp19 is important for both CO(2)/bicarbonate interconversion, and the proton transfer step of catalysis.
authors.affiliation: Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Zimmerman
authors.forename: John F
authors.initials: JF
authors.lastname: Domsic
authors.forename: Chingkuang
authors.initials: C
authors.lastname: Tu
authors.forename: Arthur H
authors.initials: AH
authors.lastname: Robbins
authors.forename: Robert
authors.initials: R
authors.lastname: McKenna
authors.forename: David N
authors.initials: DN
authors.lastname: Silverman
authors.forename: James G
authors.initials: JG
authors.lastname: Ferry
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002256:Carbonic Anhydrases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017020:Methanosarcina / Q000737:chemistry / Q000201:enzymology*; D008958:Models, Molecular; D009154:Mutation; D017433:Protein Structure, Secondary; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism; D014443:Tyrosine / Q000737:chemistry* / Q000378:metabolism
chemical_list: D019843:Archaeal Proteins; D011522:Protons; D011994:Recombinant Proteins; D014443:Tyrosine; D014364:Tryptophan; D002256:Carbonic Anhydrases