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

Plastidic pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40) wa s purified to near homogeneity as judged by native PAGE with about 4% recov ery from developing seeds of Brassica campestris using (NH4)(2)SO4 fraction ation, DEAE-cellulose chromatography, gel filtration through Sepharose-CL-6 B and affinity chromatography through reactive blue Sepharose-CL-6B. The pu rified enzyme having molecular mass of about 266 kDa was quite stable and s howed a broad pH optimum between pH 6.8-7.8. Typical Michaelis-Menten kinet ics was obtained for both the substrates with K-m values of 0.13 and 0.14 m M for PEP and ADP, respectively. The enzyme could also utilize CDP, GDP or UDP as alternative nucleotide to ADP, but with lower V-max and higher K-m. The enzyme had an absolute requirement for a divalent and a monovalent cati on for activity and was inhibited by oxalate, fumarate, citrate, isocitrate and ATP, and activated by AMP, aspartate, 3-PGA, tryptophan and inorganic phosphate. ATP inhibited the enzyme competitively with respect to PEP and n on-competitively with respect to ADP. Similarly, oxalate inhibition was als o of competitive type with respect to PEP and non-competitive with respect to ADP. This inhibition by either ATP or oxalate was not due to chelation o f Mg2+, as the inhibition was not relieved on increasing Mg2+ concentration even upto 30 mM. Initial velocity and product inhibition studies demonstra ted the reaction mechanism to be compulsory ordered type. The enzyme seems to be regulated synergistically by ATP and citrate.