The kinetic properties of phosphoenolpyruvate carboxykinase of Escherichia coli.

Initial rate kinetic studies on phosphoenolpyruvate carboxykinase of Escherichia coli are consistent with either of two sequential kinetic mechanisms: rapid equilibrium random, or partially ordered. However, the kinetics of isotope exchange at chemical equilibrium allows clear discrimination in favor of a random mechanism containing a preferred pathway of substrate addition and product release. All plots of exchange rates vs. reactant concentrations leveled off at constant rates at saturating levels of substrates; since complete inhibition of any exchange was not observed, a compulsory sequence may be eliminated. High concentrations of phosphoenolpyruvate and (or) ATP repressed the oxaloacetate in equilibrium bicarbonate exchange, however, indicating that the latter pair of substrates add most favorably to the free enzyme. Exchange rates between various substrate-product pairs were not identical, ruling out a formally rapid equilibrium random mechanism with rate-limiting interconversion of the central complex. A comparison of the relative rates of the overall reaction and the ATP-dependent oxaloacetate in equilibrium bicarbonate exchange showed the latter to be much faster than net formation of oxaloacetate. The requirement for ATP in promoting this exchange may be rationalized in terms of substrate synergism, with occupation of the ATP binding site a requisite for its catalysis.