BINDING OF CARBON-DIOXIDE TO PHOSPHOENOLPYRUVATE CARBOXYKINASE DEDUCED FROM CARBON KINETIC ISOTOPE EFFECTS

Phosphoenolpyruvate carboxykinase [ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] from Chloris gayana Kunth has been purified by a combination of ammonium sulfate fractionation, ion exchange, gel filtration, and affinity chromatography on agarose-hexane-ATP. In the direction of OAA formation, the specific activity of the enzyme was 33-mu-mol/(min-mg of protein). The carbon isotope effect on carboxylation was measured by successive analysis of remaining CO2 over the course of the reaction. At 22 mM PEP and 1.3 mM MgADP, pH 7.5, the isotope effect is 1.024 +/- 0.001. When the concentration of PEP was reduced to 1 mM, the isotope effect rose to 1.034 +/- 0.004; when the concentration of MgADP was reduced to 60-mu-M, the value rose to 1.040 +/- 0.006. The variation of the carbon isotope effect on carboxylation with both substrate concentrations indicates that the enzyme operates by a random kinetic mechanism. This in turn requires that the enzyme have a binding site for substrate CO2; this is one of the first enzymes for which such a site has been demonstrated.