title: The reductive half-reaction of xanthine dehydrogenase from Rhodobacter capsulatus: the role of Glu232 in catalysis.
abstract: The kinetic properties of an E232Q variant of the xanthine dehydrogenase from Rhodobacter capsulatus have been examined to ascertain whether Glu(232) in wild-type enzyme is protonated or unprotonated in the course of catalysis at neutral pH. We find that kred, the limiting rate constant for reduction at high [xanthine], is significantly compromised in the variant, a result that is inconsistent with Glu(232) being neutral in the active site of the wild-type enzyme. A comparison of the pH dependence of both kred and kred/Kd from reductive half-reaction experiments between wild-type and enzyme and the E232Q variant suggests that the ionized Glu(232) of wild-type enzyme plays an important role in catalysis by discriminating against the monoanionic form of substrate, effectively increasing the pKa of substrate by two pH units and ensuring that at physiological pH the neutral form of substrate predominates in the Michaelis complex. A kinetic isotope study of the wild-type R. capsulatus enzyme indicates that, as previously determined for the bovine and chicken enzymes, product release is principally rate-limiting in catalysis. The disparity in rate constants for the chemical step of the reaction and product release, however, is not as great in the bacterial enzyme as compared with the vertebrate forms. The results indicate that the bacterial and bovine enzymes catalyze the chemical step of the reaction to the same degree and that the faster turnover observed with the bacterial enzyme is due to a faster rate constant for product release than is seen with the vertebrate enzyme.
authors.affiliation: Department of Biochemistry, University of California, Riverside, California 92521 and.
authors.forename: James
authors.initials: J
authors.lastname: Hall
authors.affiliation: Department of Molecular Enzymology, Institute of Biochemistry and Biology, University Potsdam, 14476 Potsdam, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Reschke
authors.affiliation: Department of Biochemistry, University of California, Riverside, California 92521 and.
authors.forename: Hongnan
authors.initials: H
authors.lastname: Cao
authors.affiliation: Department of Molecular Enzymology, Institute of Biochemistry and Biology, University Potsdam, 14476 Potsdam, Germany.
authors.forename: Silke
authors.initials: S
authors.lastname: Leimkühler
authors.affiliation: Department of Biochemistry, University of California, Riverside, California 92521 and. Electronic address: Russ.Hille@ucr.edu.
authors.forename: Russ
authors.initials: R
authors.lastname: Hille
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain; D002417:Cattle; D018360:Crystallography, X-Ray; D018698:Glutamic Acid / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008968:Molecular Conformation; D010084:Oxidation-Reduction; D016354:Rhodobacter capsulatus / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity; D019820:Xanthine / Q000737:chemistry; D014968:Xanthine Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D014969:Xanthine Oxidase / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D019820:Xanthine; D018698:Glutamic Acid; D014968:Xanthine Dehydrogenase; D014969:Xanthine Oxidase
keywords: Enzyme Kinetics; Glutamate; Glutamine; Isotope Effect; Ultraviolet-visible Spectroscopy (UV-visible Spectroscopy); Xanthine; Xanthine Dehydrogenase; Xanthine Oxidase; pH Dependence