Kinetic mechanism and regulation of ADP-glucose pyrophosphorylase from barley (Hordeum vulgare) leaves.
Barley (Hordeum vulgare, cv. Bomi) leaf ADP-glucose pyrophosphorylase (AGP) was purified to near-homogeneity, using ammonium sulfate fractionation and heat treatment as well as ion exchange, hydrophobic, and dye-ligand chromatography. The enzyme was found to be composed of two subunit types of 51 and 54 kDa, which were recognized by the respective rabbit antibodies against the small and/or large subunit of spinach leaf AGP. Substrate kinetics and product inhibition studies, carried out in the direction of ADP-glucose synthesis, suggested a sequential Iso Ordered Bi Bi kinetic mechanism for the enzyme. In addition, inhibition patterns with CrATP, a dead-end inhibitor of ATP-utilizing enzymes, were consistent with ATP (synthesis reaction) and ADP-glucose (pyrophosphorolysis reaction) binding first to different free forms of the enzyme. The AGP was potently activated by 3-phosphoglycerate (PGA) (K alpha of 5 microM) and inhibited by orthophosphate (P(i)), with the ratio of the two effectors playing a major role in modulating AGP activity. At low [PGA]/[P(i)] ratios, P(i) could reverse the activating effect of PGA. However, at the [PGA]/[Pi] ratios of 1.4, or higher, some activating effect of P(i) in the presence of PGA was observed. PGA decreased the Km values for all substrates of AGP (in both the synthesis and pyrophosphorolysis directions). Properties of the barley leaf AGP are compared to those of AGP from barley seed endosperm, which is insensitive to PGA/P(i) regulation.
Plant Molecular Biology Laboratory, NLVF, Aas, Norway.
L A
LA
Kleczkowski
P
P
Villand
J
J
Preiss
O A
OA
Olsen
D000255:Adenosine Triphosphate / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D051599:Glucose-1-Phosphate Adenylyltransferase; D005988:Glyceric Acids / Q000378:metabolism; D001467:Hordeum / Q000201:enzymology*; D007700:Kinetics; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D013379:Substrate Specificity
D005988:Glyceric Acids; D010710:Phosphates; C005156:3-phosphoglycerate; D000255:Adenosine Triphosphate; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase