Phosphoenolpyruvate synthetase from the hyperthermophilic archaeon Pyrococcus furiosis

Phosphoenolpyruvate synthetase (PpsA) was purified from the hyperthermophil ic archaeon Pyrococcus furiosus. This enzyme catalyzes the conversion of py ruvate and ATP to phosphoenolpyruvate (PEP), AMP, and phosphate and is thou ght to function in gluconeogenesis. PpsA has a subunit molecular mass of 92 kDa and contains one calcium and one phosphorus atom per subunit. The acti ve form has a molecular mass of 690 +/- 20 kDa and is assumed to be octomer ic, while approximately 30% of the protein is purified as a large (similar to1.6 MDa) complex that is not active. The apparent K-m values and catalyti c efficiencies for the substrates pyruvate and ATP (at 80 degreesC, pH 8.4) were 0.11 mM and 1.43 x 10(4) mM(-1) . s(-1) and 0.39 mM and 3.40 x 10(3) mM(-1) s(-1), respectively, Maximal activity was measured at pH 9.0 (at 80 degreesC) and at 90 degreesC (at pH 8.4). The enzyme also catalyzed the rev erse reaction, hut the catalytic efficiency with PEP was very low [k(cat)/K -m = 32 (mM . s)(-1)]. In contrast to several other nucleotide dependent en zymes from P. furiosus, PpsA has an absolute specificity for ATP as the pho sphate-donating substrate. This is the first PpsA from a nonmethanogenic ar chaeon to be biochemically characterized, Its kinetic properties are consis tent with a role in gluconeogenesis, although its relatively high cellular concentration (similar to5% of the cytoplasmic protein) suggests an additio nal function possibly related to energy spilling. It is not known whether i nterconversion between the smaller, active and larger, inactive forms of th e enzyme has any functional role.