A reactive cysteine in avian liver phosphoenolpyruvate carboxykinase.
The modification of avian phosphoenolpyruvate carboxykinase by a variety of sulfhydryl reagents leads to inhibition. The inhibition is related to the loss of 1 highly reactive cysteine residue of the 13 cysteines present in the enzyme. Inhibition by reagents which yield a mixed disulfide was rapidly reversed by thiols. Reagents specific for vicinal sulfhydryl configurations were not potent inhibitors. The cysteine-modified enzyme continues to bind Mn2+ with the same stoichiometry and dissociation constant as the native enzyme. All of the substrates also bind to thiol-modified inactive enzyme. The modification of the reactive cysteine with the spin-labeled iodoacetate derivative leads to inactive enzyme with spin label stoichiometrically incorporated. The EPR spectrum showed an immobilized spin label on the enzyme. EPR studies of the perturbation of the phosphoenolpyruvate carboxykinase-bound spin label by bound Mn2+ showed a dipolar interaction between the two spins, estimated to be 10 A apart. The perturbation of the 1/T1 and 1/T2 values of the 31P resonances of ITP by spin-labeled enzyme indicates that this portion of the nucleotide binds 8-10 A from the spin label. These results indicate that the reactive cysteine is close to but not at the active site of the enzyme. The thiol group must be free and in its reduced form for the enzyme to be active. Perhaps modification of this group prevents conformational change(s) upon ligand binding necessary for the catalytic process.
Makinen
A L
AL
Department of Chemistry, University of Notre Dame, Indiana 46556.
Nowak
T
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D000818:Animals; D001665:Binding Sites; D002645:Chickens; D003545:Cysteine; D004578:Electron Spin Resonance Spectroscopy; D007461:Iodoacetates / Q000378:metabolism; D019807:Iodoacetic Acid; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008345:Manganese / Q000378:metabolism; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000378:metabolism*; D011485:Protein Binding
D007461:Iodoacetates; D008345:Manganese; D010729:Phosphoenolpyruvate Carboxykinase (GTP); D003545:Cysteine; D019807:Iodoacetic Acid