Kinetic and isotope effect studies of maize phosphoenolpyruvate carboxylase.
Carbon isotope effects for the carbon atom arising from bicarbonate have been measured for the phosphoenolpyruvate carboxylase from maize. At pH 7.5, 25 degrees C, the isotope effect is K12/k13 = 1.0029 +/- 0.0005 in the presence of Mg2+. The isotope effect decreases with increasing pH, reaching a value of 0.9973 at pH 10.0. All these isotope effects are relative to HCO3(-) taken as the starting state. If CO2 is considered the starting state, the isotope effects are all inverse. These values suggest that the carboxylation of phosphoenolpyruvate occurs by way of a stepwise mechanism involving an enzyme-bound carboxyphosphate intermediate, with formation of the intermediate being the primary rate-determining step. Steady-state kinetics reveal that Vmax is independent of pH over the range pH 7.5-10.0 Vmax/Km (phosphoenolpyruvate) is bell shaped in the same interval. Two pKa values near 7 are observed; the first is attributed to ionization of the phosphate group of phosphoenolpyruvate and the second to an unidentified group on the enzyme. Activity of the enzyme also depends on protonation of a group on the enzyme with a pKa near 10. Several metal ions were tested as activators of phosphoenolpyruvate carboxylase. Under saturating conditions, Mg2+ and Mn2+ show equal activity but different carbon isotope effects. Co2+ has about half the activity of Mg2+ and shows an inverse carbon isotope effect.
D001639:Bicarbonates; D002247:Carbon Isotopes; D002262:Carboxy-Lyases / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010730:Phosphoenolpyruvate Carboxylase / Q000302:isolation & purification / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D003313:Zea mays / Q000201:enzymology
D001639:Bicarbonates; D002247:Carbon Isotopes; D002262:Carboxy-Lyases; D010730:Phosphoenolpyruvate Carboxylase; D008274:Magnesium