PURIFICATION, CHARACTERIZATION AND STEADY-STATE KINETIC-PROPERTIES OFCYTOSOLIC PYRUVATE-KINASE FREE OF PHOSPHOENOL PYRUVATE PHOSPHATASE-ACTIVITY FROM GERMINATING MUNG BEANS (VIGNA-RADIATA L)

Mung bean pyruvate kinase (PK) practically free from PEP-phosphatase h as been purified about 36 fold. The enzyme is irreversibly inactivated on desalting by gel filtration or dialysis (without EDTA). The inacti vation is also observed in the presence of ATP, Mg2+ or thiols but is prevented by a non-proteinous, heat stable, small molecular mass facto r present in the mung bean extract, Mung bean PK has a molecular mass of 210 kDa. It shows single exponential decay of activity at various t emperatures (-4 to 60 degrees C). The K-m of PEP and ADP are found to be 0.12 and 0.24 mM, respectively at pH 6.5, when the enzyme is satura ted with the second substrate. The K-m values for PEP and ADP are 0.05 and 0.16 mM, at pH 8.5 and 0.09 and 0.17 mM, respectively at pH 7.5. The optimum pH is 7.5. The enzyme shows an absolute requirement for Mg 2+ (K-m 0.43 mM) or Mn2+ ions (K-m 0.125 mM). Potassium ions are not e ssential but activate the-enzyme in the presence of Mg2+ or Mn2+ ions. ATP shows competitive inhibition with ADP and non-competitive with PE P. Kinetic studies at different pHs and effects of ATP suggest the for mation of a ternary complex (E.ADP.PEP) by a combination of random and compulsory ordered pathways depending on the experimental conditions.