Effects of paraoxon and ethylparathion on choline oxidase from Alcaligenes species: inhibition and denaturation.
The kinetics and thermodynamics of the effects of paraoxon (POX) and ethylparathion (EPA) on choline oxidase (ChOx) were studied. Lineweaver-Burk plots of initial velocity data showed a parallel pattern indicating uncompetitive inhibition versus choline. The inhibition constant (K(I)) obtained from the secondary plots for POX and EPA were 0.14+/-0.01 and 0.48+/-0.05 mM, respectively, suggesting that POX is a more potent inhibitor of ChOx than EPA. UV absorption was used to monitor the denaturation of ChOx by POX and EPA. A decrease in FAD fluorescence associated with the interaction of POX and EPA with ChOx suggested a tertiary structural change. Interaction of the enzyme molecule with POX or EPA resulted in inhibition and subsequently denaturation of the enzyme. The results indicate that inhibition and denaturation of the enzyme by POX and EPA are linked, but not parallel events, with inhibition occurring at lower concentrations with respect to denaturation. This suggests that the loss of initial velocity of the enzyme is an active site specific effect and not due to global conformational changes induced by the inhibitors.
Tavakoli
H
H
Department of Biophysics, Institute of Biochemistry and Biophysics, University of Tehran, Enquelab Avenue, PO Box 13145-1384, Tehran 1384, Iran.
Ghourchian
H
H
Moosavi-Movahedi
A A
AA
Chilaka
F C
FC
D000421:Alcaligenes / Q000201:enzymology*; D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive; D002384:Catalysis; D002800:Cholinesterase Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D007306:Insecticides / Q000494:pharmacology; D007700:Kinetics; D008956:Models, Chemical; D008968:Molecular Conformation; D010261:Paraoxon / Q000494:pharmacology*; D010278:Parathion / Q000494:pharmacology*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013816:Thermodynamics; D014466:Ultraviolet Rays
D002800:Cholinesterase Inhibitors; D007306:Insecticides; D010278:Parathion; D000429:Alcohol Oxidoreductases; C019888:choline oxidase; D010261:Paraoxon