Characterization of the Family I inorganic pyrophosphatase from Pyrococcus horikoshii OT3

SUNG-JONG JEON ； KAZUHIKO ISHIKAWA

《Archaea》 2005卷 (2005/12) Pp. 385-389

https://doi.org/10.1155/2005/591628

摘要

A gene encoding for a putative Family inorganic pyrophosphatase (PPase, EC 3.6.1.1) from the hyperthermophilic archaeon Pyrococcus horikoshiiOT3was 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 °C and 7.5, respectively. The half-life of heat inactivation was about 50 min at 105 °C. The heat stability of PhPPase was enhanced in the presence of Mg^(2+).Adivalent cation was absolutely required for enzyme activity, Mg^(2+) being most effective; Zn^(2+), Co^(2+) and Mn^(2+) efficiently supported hydrolytic activity in a narrow range of concentrations (0.05-0.5 mM). The K_m for pyrophosphate and Mg^(2+) were 113 and 303 μM, respectively; and maximum velocity, V_(max), was estimated at 930 U mg^(-1).

關鍵字

archaea ； hyperthermophile