Physical and catalytic properties of pyruvate kinase from pig dental pulp.

By ammonium-sulphate fractionation, phosphocellulose column chromatography, and isoelectric focusing, pyruvate kinase (EC 2.7.1.40) was purified from pig dental pulps to yield a homogeneous preparation with a single protein band of approx. 63,000 daltons upon SDS-polyacrylamide gel electrophoresis. As the molecular weight of the enzyme was estimated to be 250,000 by Sephadex G-200 gel filtration, a tetrameric structure is indicated. The isoelectric point of the purified enzyme was 8.0; the optimal pH, pH 7.2. At this pH, there was no substrate inhibition of the enzyme by either phosphoenolpyruvate (PEP) or ADP, and the relationship between reaction velocity and each substrate concentration was well-explained by the Michaelis-Menten equation. The Km values were 53 and 286 microM for PEP and ADP, respectively. The enzyme had different catalytic properties at pH 8.0: with 0.4 mM ADP, the kinetic behaviour gave a sigmoidal curve with respect to PEP and fructose-1,6-diphosphate acted as an allosteric activator. With 2.0 mM ADP, a hyperbolic curve resulted with PEP.