title: An affinity label for the regulatory dithiol of ribulose-5-phosphate kinase from maize (Zea mays).
abstract: Ribulose-5-phosphate kinase from maize (Zea mays) can exist in either a reduced, active form or an oxidized, inactive form. Reduced ribulose-5-phosphate kinase is rapidly and irreversibly inactivated by the dichlorotriazine dye Reactive Red 1 (Procion Red MX-2B), but the irreversible inactivation of the oxidized form of ribulose-5-phosphate kinase occurs at only 0.05% of this rate. The rate of inactivation of the reduced enzyme by Reactive Red 1 (apparent bimolecular rate constant 10(4)M-1 X s-1 at pH 7.4 and 25 degrees C) is several orders of magnitude greater than previous estimates of the rates of dye-mediated inactivation of other enzymes. The dye-dependent inactivation of the reduced enzyme is inhibited by Hg2+ or p-mercuribenzoate (thiol reagents that reversibly inhibit ribulose-5-phosphate kinase activity), or by ATP and ADP, the nucleotide substrates of the enzyme. Hydrolysed Reactive Red 1, which does not inactivate the enzyme, is a reversible inhibitor of ribulose-5-phosphate kinase. This inhibition is competitive with respect to ATP (Ki approximately 0.5 mM). The dye appears to act as an affinity label for the ATP/ADP-binding site by preferentially arylating a thiol residue generated during the reductive activation of the enzyme that is achieved by dithiothreitol or thioredoxin in vitro or during illumination of leaves.
mesh_terms: D000227:Adenine Nucleotides / Q000494:pharmacology; D000345:Affinity Labels; D001665:Binding Sites; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D010084:Oxidation-Reduction; D010770:Phosphotransferases / Q000037:antagonists & inhibitors*; D017853:Phosphotransferases (Alcohol Group Acceptor); D010944:Plants / Q000201:enzymology*; D014227:Triazines / Q000494:pharmacology*; D003313:Zea mays / Q000201:enzymology
chemical_list: D000227:Adenine Nucleotides; D000345:Affinity Labels; D014227:Triazines; C003213:Procion Brilliant Red M-2BS; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase; D005033:Ethylmaleimide; D004229:Dithiothreitol