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)

Abstract

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.

Key Words:

• Electron transferring flavoprotein
• Human
• Chemical modification
• 5,5′-dithiobis-(2-nitrobenzoic acid)