Purification and characterization of cytosolic pyruvate kinase from banana fruit.
Cytosolic pyruvate kinase (PK(c)) from ripened banana (Musa cavendishii L.) fruits has been purified 543-fold to electrophoretic homogeneity and a final specific activity of 59.7 micromol of pyruvate produced/min per mg of protein. SDS/PAGE and gel-filtration FPLC of the final preparation indicated that this enzyme exists as a 240 kDa homotetramer composed of subunits of 57 kDa. Although the enzyme displayed a pH optimum of 6.9, optimal efficiency in substrate utilization [in terms of V(max)/K(m) for phosphoenolpyruvate (PEP) or ADP] was equivalent at pH 6.9 and 7.5. PK(c) activity was absolutely dependent upon the presence of a bivalent and a univalent cation, with Mg(2+) and K(+) respectively fulfilling this requirement. Hyperbolic saturation kinetics were observed for the binding of PEP, ADP, Mg(2+) and K(+) (K(m) values of 0.098, 0.12, 0.27 and 0.91 mM respectively). Although the enzyme utilized UDP, IDP, GDP and CDP as alternative nucleotides, ADP was the preferred substrate. L-Glutamate and MgATP were the most effective inhibitors, whereas L-aspartate functioned as an activator by reversing the inhibition of PK(c) by L-glutamate. The allosteric features of banana PK(c) are compared with those of banana PEP carboxylase [Law and Plaxton (1995) Biochem. J. 307, 807-816]. A model is presented which highlights the roles of cytosolic pH, MgATP, L-glutamate and L-aspartate in the co-ordinate control of the PEP branchpoint in ripening bananas.
Turner
W L
WL
Department of Biology, Queen's University, Kingston, Ontario, Canada.
Plaxton
W C
WC
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000494:Allosteric Regulation / Q000187:drug effects; D001224:Aspartic Acid / Q000494:pharmacology; D002412:Cations / Q000378:metabolism; D003067:Coenzymes / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology*; D005638:Fruit / Q000201:enzymology* / Q000254:growth & development; D018698:Glutamic Acid / Q000494:pharmacology; D005988:Glyceric Acids / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000378:metabolism; D008970:Molecular Weight; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D010730:Phosphoenolpyruvate Carboxylase / Q000378:metabolism; D011770:Pyruvate Kinase / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism*; D013816:Thermodynamics; D019862:Zingiberales / Q000201:enzymology* / Q000254:growth & development
D002412:Cations; D003067:Coenzymes; D005988:Glyceric Acids; D008670:Metals; D001224:Aspartic Acid; D018698:Glutamic Acid; D000244:Adenosine Diphosphate; D010728:Phosphoenolpyruvate; D011770:Pyruvate Kinase; D010730:Phosphoenolpyruvate Carboxylase