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REGULATION OF MAIZE LEAF PYRUVATE, ORTHOPHOSPHATE DIKINASE BY REVERSIBLE PHOSPHORYLATION (ENZYME, PROTEIN)
Abstract
The enzyme pyruvate, Pi dikinase from maize leaves is active when the plant is exposed to light and inactive when in the dark. Until recently, the molecular mechanism for this reversible light/dark regulation was unknown in spite of this enzyme's key and possibly rate-limiting role in C(,4) photosynthesis. The findings described herein document the light/dark regulation of maize leaf dikinase activity by reversible phosphorylation via complementary studies in vitro, in situ and in vivo. Experiments in vitro demonstrated that regulatory phosphorylation occurs at a threonyl residue with the (beta)-phosphate of ADP being the specific group transferred. These observations were confirmed in situ with intact maize mesophyll protoplasts and in vivo with attached leaves. Sequencing of the regulatory site peptide demonstrated that the regulatory threonyl residue is situated two amino acids away from a catalytically essential histidyl residue. The protein-substrate specificity of the maize leaf ADP:protein phosphotransferase (regulatory protein) was studied in terms of its relative ability to inactivate/phosphorylate pyruvate, Pi dikinase from Zea mays and the nonsulfur purple photosynthetic bacterium Rhodospirillum rubrum. The dimeric bacterial dikinase was inactivated by the maize leaf regulatory protein via phosphorylation, with a stoichiometry of (TURN)1 mol phosphate incorporated per mol protomer. The requirements for inactivation/phosphorylation in this heterologous system were identical to those established for the tetrameric maize leaf enzyme. The ADP-dependent maize leaf regulatory protein did not phosphorylate alternate protein-substrates such as casein or phosvitin, and its activity was not affected by cyclic nucleotides, Ca('2+) or calmodulin. The regulation of the maize leaf ADP:protein phosphotransferase was studied in terms of changes in adenylate energy charge versus pyruvate concentration. The change in adenylate energy charge necessary to substantially inhibit phosphorylation was not suggestive of it being a physiological modulator of phosphotransferase activity. Pyruvate was a potent competitive inhibitor of inactivation consistent with its interaction with the catalytic intermediate of dikinase, the true protein-substrate for ADP-dependent phosphorylation/inactivation.
Subject Area
Biochemistry
Recommended Citation
BUDDE, RAYMOND JOSEPH A, "REGULATION OF MAIZE LEAF PYRUVATE, ORTHOPHOSPHATE DIKINASE BY REVERSIBLE PHOSPHORYLATION (ENZYME, PROTEIN)" (1985). ETD collection for University of Nebraska-Lincoln. AAI8602924.
https://digitalcommons.unl.edu/dissertations/AAI8602924