title: Halophilic mechanism of the enzymatic function of a moderately halophilic dihydrofolate reductase from Haloarcula japonica strain TR-1.
abstract: Dihydrofolate (DHF) reductase coded by a plasmid of the extremely halophilic archaeon Haloarcula japonica strain TR-1 (HjDHFR P1) shows moderate halophilicity on enzymatic activity at pH 6.0, although there is no significant effect of NaCl on its secondary structure. To elucidate the salt-activation and -inactivation mechanisms of this enzyme, we investigated the effects of pH and salt concentration, deuterium isotope effect, steady-state kinetics, and rapid-phase ligand-binding kinetics. Enzyme activity was increased eightfold by the addition of 500 mM NaCl at pH 6.0, fourfold by 250 mM at pH 8.0, and became independent of salt concentration at pH 10.0. Full isotope effects observed at pH 10.0 under 0-1000 mM NaCl indicated that the rate of hydride transfer, which was the rate-determining step at the basic pH region, was independent of salt concentration. Conversely, rapid-phase ligand-binding experiments showed that the amplitude of the DHF-binding reaction increased and the tetrahydrofolate (THF)-releasing rate decreased with increasing NaCl concentration. These results suggested that the salt-activation mechanism of HjDHFR P1 is via the population change of the anion-unbound and anion-bound conformers, which are binding-incompetent and -competent conformations for DHF, respectively, while that of salt inactivation is via deceleration of the THF-releasing rate, which is the rate-determining step at the neutral pH region.
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
authors.forename: Yurina
authors.initials: Y
authors.lastname: Miyashita
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Hiroshima, 739-8526, Japan. ohmae@hiroshima-u.ac.jp.
authors.forename: Eiji
authors.initials: E
authors.lastname: Ohmae
authors.affiliation: Department of Structural Biology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Tokyo, 113-8510, Japan.
authors.forename: Teikichi
authors.initials: T
authors.lastname: Ikura
authors.affiliation: Department of Biotechnology and Chemistry, School of Engineering, Kinki University, Higashi-Hiroshima, Hiroshima, 739-2116, Japan.
authors.forename: Kaoru
authors.initials: K
authors.lastname: Nakasone
authors.affiliation: Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Hiroshima, 739-8526, Japan.
authors.forename: Katsuo
authors.initials: K
authors.lastname: Katayanagi
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry / Q000378:metabolism*; D019613:Haloarcula / Q000201:enzymology*; D007700:Kinetics; D011485:Protein Binding; D054712:Salinity; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; D013762:Tetrahydrofolate Dehydrogenase
keywords: Dihydrofolate reductase; Haloarcula japonica; Halophilic enzyme; Salt activation; Salt inactivation