Regulatory phosphorylation of banana fruit phosphoenolpyruvate carboxylase by a copurifying phosphoenolpyruvate carboxylase-kinase.
Phosphoenolpyruvate (P-pyruvate) carboxylase from ripened banana fruit was purified to near homogeneity and a final specific activity of 20-23 U/mg protein; P-pyruvate carboxylase-kinase copurified with P-pyruvate carboxylase throughout the purification. Gel filtration FPLC indicated that the two proteins form a tightly associated 425-kDa complex. Both the 103-kDa and 100-kDa subunits of the P-pyruvate carboxylase alpha2beta2 hetetrotetramer were phosphorylated and subsequently dephosphorylated in vitro in a time-dependent manner when the final preparation was incubated with 0.1 mM [gamma-32P]ATP followed by rabbit muscle protein phosphatase type 2A1. Phosphoamino acid and phosphopeptide map analyses indicated that in vitro phosphorylation of both subunits likely occurs at an identical Ser residue. Maximal stoichiometry of 32P incorporation was 0.2 and 0.4 mol/mol 103-kDa and 100-kDa subunit, respectively. The level of 32P incorporation was correlated with the enzyme's activation state when assayed under suboptimal assay conditions (pH 7.3, 75 microM P-pyruvate, 0.2 mM L-malate). The main kinetic effect of phosphorylation was to decrease the enzyme's Km(P-pyruvate), as well as its sensitivity to inhibition by L-malate and L-glutamate. Banana P-pyruvate carboxylase-kinase: (a) also phosphorylated maize leaf P-pyruvate carboxylase, histone III-S, and dephosphorylated casein; (b) demonstrated Mg2+ dependence and Ca2+ independence, (c) exhibited a broad pH activity optimum of pH 8.0-8.5, and (d) was inhibited by L-malate and activated by Ba2+ and Co2+. Time-course kinetic studies suggested that P-pyruvate carboxylase exists mainly in the dephosphorylated form in preclimacteric, climacteric and postclimacteric fruit, but that its kinase is expressed throughout ripening. In situ 32P-labeling indicated that, while both subunits of ripe banana P-pyruvate carboxylase are phosphorylated in vivo, it is primarily the 100-kDa subunit that is radiolabeled. The results suggest that similar to the enzyme from leaves, root nodules and seeds, a fruit P-pyruvate carboxylase may be subject to regulatory seryl phosphorylation by an endogenous P-pyruvate carboxylase-kinase.
Law
R D
RD
Department of Biology, Queen's University, Kingston, Ontario, Canada.
Plaxton
W C
WC
D000818:Animals; D002850:Chromatography, Gel; D005638:Fruit / Q000201:enzymology*; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000201:enzymology; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000302:isolation & purification / Q000378:metabolism*; D010730:Phosphoenolpyruvate Carboxylase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D018515:Plant Leaves; D011485:Protein Binding; D011817:Rabbits; D013379:Substrate Specificity; D003313:Zea mays / Q000201:enzymology
D046911:Macromolecular Substances; D010748:Phosphopeptides; D010768:Phosphoserine; D010749:Phosphoprotein Phosphatases; D010730:Phosphoenolpyruvate Carboxylase; D010729:Phosphoenolpyruvate Carboxykinase (GTP)