Stabilization, partial purification, and effects of activating cations, ADP, and phosphoenolpyruvate on the reaction rates of an erythrocyte pyruvate kinase

Abstract

Stability of erythrocyte pyruvate kinase was found to be independently enhanced by inorganic ortho-phosphate, magnesium, and 2-mercaptoethanol. Phosphoenolpyruvate could substitute for and was more effective than phosphate. The enzyme was more stable below pH 7.4 than above. These data were utilized to help achieve a partial purification (<10,000-fold), of the enzyme. The purified enzyme had a pH optimum of 6.8 – 7.1, was activated by potassium (or ammonium), and also required magnesium for activity. The activating effect of magnesium was proportional to the amount of magnesium-ADP chelate formed. Excess magnesium acted as an inhibitor, competing with potassium. The K m value for magnesium-ADP was found to be about 3.3 × 10 −4 m. High levels of ADP were found to inhibit reaction rates. This inhibition was released by addition of more magnesium, suggesting free ADP was the inhibitor. The K m value for phosphoenolpyruvate was found to be about 4.6 × 10 −5 m. Optimal assay conditions (TES buffer) were found to be at about: pH 7.0, 90 m m K; 6.4 m m Mg: 2 m m ADP, and 0.8 m m PEP.