Kinetic studies were done to obtain a quantitative estimation of the synergistic interactions that occur between phosphoenolpyruvate carboxylase (orthophosphate:oxaloacetate carboxylase (phosphorylating) E.C. 188.8.131.52) from Escherichia coCi K12 and various combinations of its primary substrate, P-enolpyruvate, and the activators acetylcoenzyme A, CDP, GTP, and fructose 1,6-bisphosphate. The analysis involves the evaluation of apparent K values, Ks for P-enolpyruvate and KA for activators, as a… CONTINUE READING
FIG. 3. Graphical evaluation of the dissociation of P-enolpyruvate and acetylcoenzyme A from the ternary enzyme complex. Apparent K values for P-enolpyruvate (top panel) and for acetyl coenzyme A (bottom panel) are plotted against acetyl coenzyme A and P-enolpyruvate concentrations, respectively, in accordance with Equation 5. The slope intercept ratio from the top graph is a measure of the dissociation constant of acetyl coenzyme A from the ternary complex, EBS = ES + B, and the same ratio from the bottom graph is a measure of the P-enolpyruvate dissociation constant from the same complex, EBS = EB + S. The K I and KZ values determined as described under "Materials and Methods" are 60.6 mM and 0.95 mM for P-enolpyruvate and acetyl coenzyme A dissociation from the binary ES and EB complexes, respectively. Apparent K values were determined from saturation curves covering at least a 10-fold range in concentration with at least six data points. Solid triangles (A) and solid circles (0) represent separate experiments. Error bars on the measured values are shown by the vertical lines. Error bars are omitted if the estimated error was less than the diameter of the points.