Properties and reaction mechanism of C₄ leaf pyruvate,P_i dikinase

The properties and reaction mechanism of maize leaf pyruvate,P_i dikinase are described. K_m values were determined for the forward reaction substrates, pyruvate, ATP, and P_i, at pH 7.4 and 8.0 and for reverse reaction substrates at pH 7.4. Enzyme activity was almost totally dependent on added monovalent cations in both directions. NH₄⁺ was most effective, with K_a values of about 0.38 mm for the forward reaction and 2 mm for the reverse reaction. K⁺ also completely activated the enzyme in the forward direction (K_a = 8 mM) but only partially activated in the reverse direction. Na⁺ had little effect on either reaction. The pH optimum for the forward reaction was about 8.2; the reverse reaction optimum was about 6.9. Maximum activity for the reverse direction was about twice the maximum forward direction rate. From data on the requirements for the ATP-AMP exchange reaction, on the mechanism of inhibition of the forward reaction by PEP, AMP, and PP_i and from the kinetics of the interaction of varying certain substrate pairs, it was concluded that the maize leaf pyruvate,P_i dikinase reaction proceeded by the two-step Bi Bi Uni Uni mechanism. This differs from the mechanism of catalysis by the bacterial enzyme.