Purification and characterization of cytosolic pyruvate kinase from developing seeds of Brassica campestris L.

Cytosolic pyruvate kinase (ATP: Pyruvate phosphotransferase, EC 2.7.1.40; P Kc) was purified to apparent homogeneity with about 22% recovery from devel oping seeds of Brassica campestris using (NH4)(2)SO4 fractionation, DEAE-ce llulose chromatography, gel filtration through Sepharose-CL-6B and affinity chromatography through reactive Blue Sepharose-CL-6B, The purified enzyme with molecular mass of about 214 kDa was a heterotetramer with subunit mole cular mass of 55 and 57 kDa. The enzyme showed maximum activity at pH 6.8 a nd absolute requirement for a divalent (Mg2+) and a monovalent (K+) cation for activity. Typical Michaelis- Menten kinetics was obtained for both the substrates with K-m values of 0.10 and 0.11 mM for PEP and ADP, respectivel y. The enzyme could also use UDP or GDP as alternative nucleotides, but wit h lower V-max and lesser affinities. The enzyme was inhibited by glutamate, glutamine, fumarate, citrate, isocitrate, oxalate, 2-PGA, ATP, UTP and GTP and activated by glucose-6-phosphate, fructose-1,6-bisphosphate and Pi, su ggesting its regulation mainly by TCA cycle intermediates and the cellular need for carbon skeletons for amino acid biosynthesis. ATP inhibition was o f competitive type with respect to PEP and non-competitive with respect to ADP. Similarly, oxalate inhibition was also of competitive type with respec t to PEP and non-competitive with respect to ADP. Initial velocity and prod uct inhibition studies except for pyruvate inhibition were consistent for a compulsory-ordered tri-bi mechanism.