Redox active sulfhydryls are required for fructose 2,6-bisphosphate activation of plant pyrophosphate fructose-6-phosphate 1-phosphotransferase.
The classical, alpha/beta-subunit form (Q2) of green tomato pyrophosphate fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90), a cytosolic enzyme functional in carbohydrate metabolism, was rapidly inactivated on incubation with the oxidant 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Analysis of the DTNB-treated sample by a fluorescence procedure revealed that inactivation was accompanied by oxidation of sulfhydryl groups, primarily on the alpha-subunit. Phosphate metabolites--fructose 2,6-bisphosphate, fructose 1,6-bisphosphate, Pi, and PPi--protected against DTNB inactivation to varying degrees. The Km values for fructose 6-phosphate and PPi were not changed by DTNB treatment, but the capability for activation by fructose 2,6-bisphosphate was severely diminished. The oxidative inactivation of PFP was reversed by dithiothreitol, but not by monothiols (reduced glutathione or beta-mercaptoethanol). Reactivation was accompanied by restoration of the ability to undergo activation by fructose 2,6-bisphosphate. The findings suggest that sulfhydryl groups are essential for the activation of PFP by fructose 2,6-bisphosphate and raise the possibility that a reversible change in their redox status can take place under certain conditions. Evidence that this is the case was obtained with a preparation from wheat flour which, in the absence of an added oxidant, required reduction by a dithiol for activation by fructose 2,6-bisphosphate (dithiothreitol and reduced thioredoxin h).
Kiss
F
F
Department of Plant Biology, University of California, Berkeley 94720.
Wu
M X
MX
Wong
J H
JH
Balogh
A
A
Buchanan
B B
BB
D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004793:Enzyme Reactivators / Q000494:pharmacology; D005635:Fructosediphosphates / Q000494:pharmacology*; D005978:Glutathione / Q000494:pharmacology; D008623:Mercaptoethanol / Q000494:pharmacology; D010084:Oxidation-Reduction; D010770:Phosphotransferases / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D004793:Enzyme Reactivators; D005635:Fructosediphosphates; D013438:Sulfhydryl Compounds; D008623:Mercaptoethanol; C027652:fructose 2,6-diphosphate; D004228:Dithionitrobenzoic Acid; D010770:Phosphotransferases; C022502:pyrophosphate-fructose 6-phosphate 1-phosphotransferase; D005978:Glutathione; D004229:Dithiothreitol