5,5'-Dithiobis-(2-nitrobenzoic acid) as a probe for a non-essential cysteine residue at the medium chain acyl-coenzyme A dehydrogenase binding site of the human 'electron transferring flavoprotein' (ETF).
Human 'electron transferring flavoprotein' (ETF) was inactivated by the thiol-specific reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB). The kinetic profile showed the reaction followed pseudo-first-order kinetics during the initial phase of inactivation. Monitoring the release of 5-thio-2-nitrobenzoate (TNB) showed that modification of 1 cysteine residue was responsible for the loss of activity. The inactivation of ETF by DTNB could be reversed upon incubation with thiol-containing reagents. The loss of activity was prevented by the inclusion of medium chain acyl-CoA dehydrogenase (MCAD) and octanoyl-CoA. Cyanolysis of the DTNB modified-ETF with KCN led to the release of TNB accompanied presumably by the formation of the thio-cyano enzyme and with almost full recovery of activity. Conservation studies and the lack of 100% inactivation, however, suggested that this cysteine residue is not essential for the interaction with MCAD.
Parker
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A
Department of Biochemistry, University College Dublin, Ireland. pengel@macollamh.ucd.ie
Engel
P C
PC
D042964:Acyl-CoA Dehydrogenase; D044944:Acyl-CoA Dehydrogenases / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D015415:Biomarkers / Q000032:analysis; D003545:Cysteine / Q000737:chemistry*; D004228:Dithionitrobenzoic Acid / Q000032:analysis*; D004579:Electron Transport; D044927:Electron-Transferring Flavoproteins; D005420:Flavoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D015335:Molecular Probes; D011485:Protein Binding; D013439:Sulfhydryl Reagents / Q000493:pharmacokinetics
D015415:Biomarkers; D044927:Electron-Transferring Flavoproteins; D005420:Flavoproteins; D015335:Molecular Probes; D013439:Sulfhydryl Reagents; D004228:Dithionitrobenzoic Acid; D044944:Acyl-CoA Dehydrogenases; D042964:Acyl-CoA Dehydrogenase; D003545:Cysteine