title: Chemical modification of the bifunctional regulatory protein of maize leaf pyruvate,orthophosphate dikinase. Evidence for two distinct active sites.
abstract: The active site(s) of the bifunctional regulatory protein of pyruvate,orthophosphate dikinase catalyze(s) the Pi-dependent activation (dephosphorylation) and ADP-dependent inactivation (phosphorylation) of maize leaf dikinase. The chemical modification studies of the regulatory protein active sites presented in this paper are interpreted as showing the two sites to be physically distinct. Pyridoxal 5'-phosphate and 2-nitro-5-thiocyanatobenzoate (NTCB) selectively inhibit the dikinase activating site, which is protected by the nonprotein substrate, Pi. Phenylglyoxal blocks both the activation and inactivation sites; the former is protected selectively by Pi and the latter by both the nonprotein substrate, ADP, and Pi. The Pi that protects the inactivation site is distinct from the activation substrate. Inhibition studies show Pi to be a parabolic competitive inhibitor of the ADP-dependent inactivation of dikinase, implying that besides substrate Pi, a second phosphate also binds to the regulatory protein. The above chemical modifications are not mutually exclusive; neither NTCB, 5,5'-dithiobis-(2-nitrobenzoate), nor pyridoxal 5'-phosphate blocks subsequent modification of the activation site by phenylglyoxal. Similarly, prior modification with NTCB does not affect modification by pyridoxal 5'-phosphate.
mesh_terms: D000244:Adenosine Diphosphate / Q000031:analogs & derivatives; D001665:Binding Sites / Q000187:drug effects; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D010658:Phenylglyoxal / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D010940:Plant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D011772:Pyruvate, Orthophosphate Dikinase / Q000378:metabolism*; D013861:Thiocyanates / Q000494:pharmacology; D003313:Zea mays / Q000201:enzymology*
chemical_list: D010710:Phosphates; D010940:Plant Proteins; D013861:Thiocyanates; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D010770:Phosphotransferases; C037396:PPDK protein, Zea mays; D011772:Pyruvate, Orthophosphate Dikinase; D004047:Diethyl Pyrocarbonate; D010658:Phenylglyoxal; C008658:2-nitro-5-thiocyanobenzoic acid