Purification and characterizaton of plastidic pyruvate kinase from developing seeds of Brassica campestris L.
Plastidic pyruvate kinase (ATP: pyruvate phosphotransferase, EC 2.7.1.40) was purified to near homogeneity as judged by native PAGE with about 4% recovery from developing seeds of Brassica campestris using (NH4)2SO4 fractionation, DEAE-cellulose chromatography, gel filtration through Sepharose-CL-6B and affinity chromatography through reactive blue Sepharose-CL-6B. The purified enzyme having molecular mass of about 266 kDa was quite stable and showed a broad pH optimum between pH 6.8-7.8. Typical Michaelis-Menten kinetics was obtained for both the substrates with K(m) values of 0.13 and 0.14 mM for PEP and ADP, respectively. The enzyme could also utilize CDP, GDP or UDP as alternative nucleotide to ADP, but with lower Vmax and higher K(m). The enzyme had an absolute requirement for a divalent and a monovalent cation 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 non-competitively with respect to ADP. Similarly, oxalate inhibition was also 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 of Mg2+, as the inhibition was not relieved on increasing Mg2+ concentration even upto 30 mM. Initial velocity and product inhibition studies demonstrated the reaction mechanism to be compulsory ordered type. The enzyme seems to be regulated synergistically by ATP and citrate.
Singh
D K
DK
Department of Chemistry and Biochemistry, CCS Haryana Agricultural University, Hisar.
Malhotra
S P
SP
Singh
R
R
D001937:Brassica / Q000737:chemistry / Q000201:enzymology*; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D005590:Fractional Precipitation; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D018087:Plastids / Q000201:enzymology*; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification*; D012639:Seeds / Q000737:chemistry; D013379:Substrate Specificity
D011770:Pyruvate Kinase