Studies on the dark/light regulation of maize leaf pyruvate, orthophosphate dikinase by reversible phosphorylation.
In maize leaves, pyruvate, orthophosphate dikinase (PPDK) is deactivated in the dark and reactivated in the light. Studies in vitro using purified PPDK and a partially purified regulatory protein from maize confirmed previous reports correlating deactivation/reactivation with the reversible phosphorylation/dephosphorylation of a threonyl residue. By monitoring the stability of the exogenous 32P-labeled adenylate substrates during deactivation, we have firmly established ADP as the specific phosphate donor. In isolated maize leaf mesophyll protoplasts preilluminated with 32Pi, we observed a three- to fivefold higher PPDK activity in situ in the light, and a corresponding three- to fivefold higher level of phosphorylation of the 94-kDa PPDK protomer in the dark. HPLC-based phosphoamino acid analysis of PPDK purified from maize leaves of both light- and dark-adapted plants revealed the presence of P-serine. The inactive enzyme from dark-adapted plants (inactivated in vivo) also contained P-threonine. Total phosphate content of PPDK purified from leaves of light-adapted plants was approximately 0.5 mol/mol protomer, and 1.5 mol/mol protomer from leaves of dark-adapted plants. Since the difference between enzyme purified from light-adapted (active PPDK) and dark-adapted (inactive PPDK) plants is the presence of P-threonine in the latter, this suggests an inactivation stoichiometry in vivo of 1 mol P-threonine/mol 94-kDa protomer. These complementary studies with maize leaf PPDK in vitro, in situ, and in vivo provide convincing evidence for the dark/light regulation of this key C4-photosynthesis enzyme by reversible phosphorylation.
D000244:Adenosine Diphosphate / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003624:Darkness; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008027:Light; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D011772:Pyruvate, Orthophosphate Dikinase / Q000378:metabolism*; D003313:Zea mays / Q000201:enzymology
D000244:Adenosine Diphosphate; D010770:Phosphotransferases; D011772:Pyruvate, Orthophosphate Dikinase