Purification, characterisation and steady state kinetic properties of cytosolic 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 has been purified about 36 fold. The enzyme is irreversibly inactivated on desalting by gel filtration or dialysis (without EDTA). The inactivation is also observed in the presence of ATP, Mg2+ or thiols but is prevented by a non-proteinous, heat stable, small molecular mass factor 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 temperatures (-4 to 60 degrees C). The Km of PEP and ADP are found to be 0.12 and 0.24 mM, respectively at pH 6.5, when the enzyme is saturated with the second substrate. The Km 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 Mg2+ (Km 0.43 mM) or Mn2+ ions (Km 0.125 mM). Potassium ions are not essential but activate the enzyme in the presence of Mg2+ or Mn2+ ions. ATP shows competitive inhibition with ADP and non-competitive with PEP. Kinetic studies at different pHs and effects of ATP suggest the formation of a ternary complex (E.ADP.PEP) by a combination of random and compulsory ordered pathways depending on the experimental conditions.
Ambasht
P K
PK
Department of Biochemistry, Faculty of Science, Banaras Hindu University, Varanasi, India.
Malhotra
O P
OP
Kayastha
A M
AM
D000135:Acid Phosphatase / Q000302:isolation & purification*; D000645:Ammonium Sulfate; D002413:Cations, Divalent / Q000494:pharmacology; D002848:Chromatography, DEAE-Cellulose; D003600:Cytosol / Q000201:enzymology; D007887:Fabaceae / Q000201:enzymology*; D007700:Kinetics; D008970:Molecular Weight; D010946:Plants, Medicinal; D011770:Pyruvate Kinase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D012639:Seeds; D013816:Thermodynamics
D002413:Cations, Divalent; D011770:Pyruvate Kinase; C070989:phosphoenolpyruvate phosphatase; D000135:Acid Phosphatase; D000645:Ammonium Sulfate