Fig 2. Double reciprocal plots from the initial velocity data of the TpPK reaction.

The reaction medium consisted of 3 ml of 50 mM Tris-HCl pH 6.0 containing 0.24 mM NADH, 30 mM Mg²⁺ _free, and 8 μg/ml LDH. The reciprocals of the concentrations of ionized PEP and ADP-Mg complexes are shown in the abscissas of each graph. A. The fixed concentrations of ADP-Mg were 0.084 (■), 0.12 (●), 0.16 (▲), 0.37 (▼), and 0.67 mM (♦). B. The fixed concentrations of PEP^3- were 0.031 (■), 0.14 (●), 0.34 (▲), 0.69(▼), and 1.30 mM (♦). The Mg²⁺ _free concentration was kept constant at 30 mM. To maintain the ionic strength, (CH₃)₄NCl was added to a final salt concentration of 0.25 M. The reaction was started with the addition of PK. The concentrations of PK were 0.32 and 0.16 μg/ml for the three lowest and two highest substrate concentrations, respectively.