The kinetic mechanism of phosphoenolpyruvate carboxykinase from Panicum maximum

The kinetic properties of phosphoenolpyruvate carboxykinase [ATP: oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49] from leaves of the C4 grass Panicum maximum have been re-evaluated due to conflicting reports concerning pH optima, Mn²⁺ requirement of activity and K_m for different substrates among C4 and CAM plants. The native enzyme has a M_r of 360 000 and a strict requirement of Mn²⁺ for activity. The optimum pH for the carboxylation reaction is 6.8 and the K_m's for PEP, ADP and HCO₃ ^- are 1.46 mM, 60 μM and 3.7 mM, respectively; for the decarboxylation process the pH optimum is 8.4 and the respective K_m for ATP and oxaloacetate are 21 and 61 μM. At pH 7.6 the maximum rate of the decarboxylation reaction is three-fold higher than that of the carboxylation process. Steady-state kinetic studies of the decarboxylation reaction under optimal conditions and the evaluation of product inhibition effects show that the kinetic mechanism of this enzyme is a sequential ordered bi-tri process, ATP being the first substrate entering the active site, followed by oxaloacetate, while CO₂ is the first substrate to emerge, followed by PEP and ADP.