Values For Phosphoenolpyruvate Research Articles

Regulation of phosphoenolpyruvate carboxylase of Zea mays by metabolites.

Crude preparations of phosphoenolpyruvate carboxylase obtained from aetiolated seedlings of Zea mays are unstable but can be stabilized with glycerol. At the pH optimum of 8.3, the K(m) value for phosphoenolpyruvate is 80mum. When assayed at 30 degrees C, the enzyme shows normal Michaelis-Menten kinetics, but when assayed at 45 degrees C sigmoid kinetics are exhibited. At pH7.0 the enzyme is inhibited by a number of dicarboxylic acids and by glutamate and aspartate. d and l forms of the hydroxy acids and amino acids are inhibitory and the kinetics approximate to simple non-competitive inhibition. The same compounds produce less inhibition at pH7.6 than at pH7.0 and the kinetics of inhibition are more complex. The enzyme is activated by P(i), by SO(4) (2-) and by a number of sugar phosphates. Maximum activation occurs at acid pH values, where enzyme activity is lowest. The enzyme is activated by AMP and inhibited by ADP and ATP so that the response to energy charge is of the R type and is thus at variance with Atkinson's (1968) concept of energy charge. The physiological significance of the response to metabolites is discussed.

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

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.

The Enzymatic Carboxylation of Phosphoenolpyruvate: I. PURIFICATION AND PROPERTIES OF PHOSPHOENOLPYRUVATE CARBOXYLASE

Abstract Phosphoenolpyruvate carboxylase has been isolated from germinating peanut cotyledons and purified 2700-fold. The purified enzyme is approximately 80% pure as judged from sedimentation patterns, has a sedimentation coefficient (s20,w) of 13.9 S, and catalyzes the carboxylation of 50 µmoles of phosphoenolpyruvate per min per mg of protein (refractometrically determined) at 30°. Enzymatic carboxylation occurs optimally at pH 8.0 to 8.2. The Km values determined for HCO3-, phosphoenolpyruvate, and Mg2+ are 3.1 x 10-4, 5 to 6 x 10-4, and 3 to 4 x 10-4 m, respectively, at pH 7.9. Studies on the effect of pH on the Km values for phosphoenolpyruvate and Mg2+ were conducted. Evaluation of plots of -log Km (Mg2+ and phosphoenolpyruvate) against pH indicate that a dissociable group (or groups) of the carboxylase having a pK of 7.3, presumably an imidazole group, is involved in Mg2+ and phosphoenolpyruvate binding. Other evidence supporting this view is presented. During the enzymatic carboxylation of phosphoenolpyruvate, 18O from substrate HC18O3- is incorporated into the orthophosphate and oxalacetate reaction products in a ratio of 1:2. This indicates that the HCO3- (or CO32-) anion rather than CO2 is the active species in the carboxylation reaction. A mechanism is proposed which is consistent with this and other observations on the phosphoenolpyruvate carboxylase-catalyzed reaction.