Plant cytosolic pyruvate kinase: a kinetic study.
The kinetic properties of cytosolic pyruvate kinase (PKc) from germinating castor oil seeds (COS) have been investigated. From experiments in which the free Mg2+ concentration was varied at constant levels of either the complexed or free forms of the substrates it was determined that the true substrates are the free forms of both phosphoenolpyruvate (PEP) and ADP. This conclusion is corroborated by the quenching of intrinsic PKC tryptophan fluorescence by free PEP and ADP. Mg2+ is bound as the free bivalent cation but is likely released as MgATP. The fluorescence data, substrate interaction kinetics, and pattern of inhibition by products and substrate analogues (adenosine 5'-O-(2-thiodiphosphate) for ADP and phenyl phosphate for PEP) are compatible with a sequential, compulsory-ordered, Tri-Bi type kinetic reaction mechanism. PEP is the leading substrate, and pyruvate the last product to abandon the enzyme. The dissociation constant and limiting Km for free PEP (8.2 to 22 and 38 microM, respectively) and the limiting Km for free ADP (2.9 microM) are considerably lower than those reported for the non-plant enzyme. The results indicate that COS PKc exists naturally in an activated state, similar to the fructose 1,6-bisphosphate-activated yeast enzyme. This deduction is consistent with a previous study (F.E. Podestá and W.C. Plaxton (1991) Biochem. J. 279, 495-501) that failed to identify any allosteric activators for the COS PKc, but which proposed a regulatory mechanism based upon ATP levels and pH-dependent alterations in the enzyme's response to various metabolite inhibitors. As plant phosphofructokinases display potent inhibition by PEP, the overall rate of glycolytic flux from hexose 6-phosphate to pyruvate in the plant cytosol will ultimately depend upon variations in PEP levels brought about by the regulation of PKc.
Podestá
F E
FE
Department of Biology, Queen's University, Kingston, Ontario, Canada.
Plaxton
W C
WC
D003600:Cytosol / Q000201:enzymology*; D006019:Glycolysis; D007700:Kinetics; D010944:Plants / Q000201:enzymology*; D011770:Pyruvate Kinase / Q000737:chemistry*; D012639:Seeds / Q000201:enzymology
D011770:Pyruvate Kinase