Role of Metabolites in the Reversible Light Activation of Pyruvate, Orthophosphate Dikinase in Zea mays Mesophyll Cells in Vivo

Abstract

Whole leaf and mesophyll cell concentrations of pyruvate, phosphoenolpyruvate (PEP), ATP, and ADP were determined in Zea mays during the reversible light activation of pyruvate, orthophosphate dikinase in vivo. Mesophyll cell levels of the four metabolites were estimated by extrapolation from values in freeze-quenched leaf samples that were fractionated by differential filtration through nylon mesh nets (adapted from M Stitt, HW Heldt [1985] Planta 164: 179-188). During the 3 minutes required for complete light activation of dikinase, pyruvate levels in the mesophyll cell decreased (from 166 ± 15 to 64 ± 10 nanomoles per milligram of chlorophyll [nmol/mg Chi]) while PEP levels increased (from 31 ± 4 to 68 ± 4 nmol/mg Chi, with a transient burst of 133 ± 16 nmol/mg Chi at 1 minute). Mesophyll cell levels of ATP increased (from 22 ± 4 to 48 ± 3 nmol/mg Chi) and ADP levels decreased (from 16 ± 4 to 7 ± 6 nmol/mg Chl) during the first minute of illumination. Upon darkening of the leaf and inactivation of dikinase, pyruvate levels initially increased in the mesophyll (from 160 ± 30 to a maximum of 625 ± 40 nmol/ mg Chi), and then slowly decreased to about the initial value in the light over an hour. PEP levels dropped (from 176 ± 5 to 47 ± 3 nmol/mg Chi) in the first 3 minutes and remained low for the remainder of the dark period. Mesophyll levels of ATP and ADP rapidly decreased and increased, respectively, about twofold upon darkening. The trends observed for these metabolite levels in the mesophyll cell during the light/dark regulation of pyruvate,orthophosphate dikinase activity suggest that pyruvate and PEP do not play a major role in vivo in regulating the extent of light activation (dephosphorylation) or dark inactivation (ADPdependent threonyl phosphorylation) of dikinase by its bifunctional regulatory protein. While the changes in ADP levels appear qualitatively consistent with a regulatory role for this metabolite in the light activation and dark inactivation of dikinase, they are not of a sufficient magnitude to account completely for the tenfold change in enzyme activity observed in vivo.Pyruvate,orthophosphate dikinase (EC 2.7.9.1) catalyzes the conversion of pyruvate, ATP and Pi to PEP,3 AMP, and

Posttranslational Modification of Maize Chloroplast Pyruvate Orthophosphate Dikinase Reveals the Precise Regulatory Mechanism of Its Enzymatic Activity

“…These studies confirmed that PDRP is a Ser/Thr kinase that requires a phosphorylated His in the target enzyme (Burnell and Hatch, 1986). This regulatory threonyl phosphorylation of PPDK is a monocyclic cascade (Stadtman and Chock, 1977) in which the covalent modification system is assumed to be a continuous process that allows the extent of PPDK activation to be attuned to the metabolic needs (Roeske and Chollet, 1989). Therefore, PDRP can alter the activation state of its target enzyme, PPDK, according to the concentrations of metabolites (e.g.…”

Pyruvate,Orthophosphate Dikinase in Leaves and Chloroplasts of C3 Plants Undergoes Light-/Dark-Induced Reversible Phosphorylation

“…1; Burnell and Hatch, 1985;Roeske and Chollet, 1989;Ashton et al, 1990). These same studies have not yielded a conclusive understanding of exactly how its opposing protein kinase and phosphatase activities are regulated, but a proposed mechanism involves the balance of competing substrates for RP (primarily ADP and Pi) in the chloroplast stroma (Burnell and Hatch, 1985;Roeske and Chollet, 1989;Ashton et al, 1990, Malkin andNiyogi, 2000). According to this working model, lower levels of stromal ADP in the light favor the reactivation/dephosphorylation (PP i -forming) reaction.…”

“…Although C 4 RP has not been reproducibly purified to homogeneity (compare Burnell and Hatch, 1985;Roeske and Chollet, 1987;Smith et al, 1994b) and therefore no related antibodies or gene clones presently exist, studies of the partially purified enzyme have elucidated a general reaction mechanism ( Fig. 1; Burnell and Hatch, 1985;Roeske and Chollet, 1989;Ashton et al, 1990). These same studies have not yielded a conclusive understanding of exactly how its opposing protein kinase and phosphatase activities are regulated, but a proposed mechanism involves the balance of competing substrates for RP (primarily ADP and Pi) in the chloroplast stroma (Burnell and Hatch, 1985;Roeske and Chollet, 1989;Ashton et al, 1990, Malkin andNiyogi, 2000).…”

“…Moreover, ADP is also a potent competitive inhibitor of the dephosphorylation reaction in vitro with respect to the inactive, PPDK-ThrP enzyme form (Burnell and Hatch, 1985;Roeske and Chollet, 1989;Ashton et al, 1990). When spinach chloroplasts were illuminated in the presence of the PSII inhibitor DCMU, light-induced dephosphorylation of phospho-PPDK was inhibited (Fig.…”

“…In this manner, the "target" enzyme's phosphorylation state is estimated by its level of activity. Using this indirect method, time course experiments measuring C 4 PPDK activation and deactivation during light/dark transitions have been published (Yamamoto et al, 1974;Nakamoto and Edwards, 1986;Roeske and Chollet, 1989;Nakamoto and Young, 1990). We performed similar kinetic studies with representative C 4 (maize) and C 3 (V. faba and F. pringlei) leaves, but directly assessed PPDK phosphorylation state during light/dark transitions.…”