title: Characterization of the Family I inorganic pyrophosphatase from Pyrococcus horikoshii OT3.
abstract: A gene encoding for a putative Family I inorganic pyrophosphatase (PPase, EC 3.6.1.1) from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 was cloned and the biochemical characteristics of the resulting recombinant protein were examined. The gene (Accession No. 1907) from P. horikoshii showed some identity with other Family I inorganic pyrophosphatases from archaea. The recombinant PPase from P. horikoshii (PhPPase) has a molecular mass of 24.5 kDa, determined by SDS-PAGE. This enzyme specifically catalyzed the hydrolysis of pyrophosphate and was sensitive to NaF. The optimum temperature and pH for PPase activity were 70 degrees C and 7.5, respectively. The half-life of heat inactivation was about 50 min at 105 degrees C. The heat stability of PhPPase was enhanced in the presence of Mg2+. A divalent cation was absolutely required for enzyme activity, Mg2+ being most effective; Zn2+, Co2+ and Mn2+ efficiently supported hydrolytic activity in a narrow range of concentrations (0.05-0.5 mM). The K(m) for pyrophosphate and Mg2+ were 113 and 303 microM, respectively; and maximum velocity, V(max), was estimated at 930 U mg(-1).
authors.affiliation: Department of Biotechnology and Bioengineering, Dong-Eui University, Busan, 614-714, Korea.
authors.forename: Sung-Jong
authors.initials: SJ
authors.lastname: Jeon
authors.forename: Kazuhiko
authors.initials: K
authors.lastname: Ishikawa
mesh_terms: D011756:Diphosphates / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D008970:Molecular Weight; D043902:Pyrococcus horikoshii / Q000201:enzymology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012969:Sodium Fluoride / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D011756:Diphosphates; D011994:Recombinant Proteins; D012969:Sodium Fluoride; D043564:Inorganic Pyrophosphatase