Reaction of both active site thiols of reduced thioredoxin reductase with N-ethylmaleimide.
Thioredoxin reductase from Escherichia coli, only in its reduced state, reacts rapidly with 2 mol of N-ethylmaleimide, which specifically alkylates both active site cysteine residues. This dual modification supports previous studies indicating that a base lowers the pK of both active site cysteine residues. The dual modification also indicates that the region around the active site dithiol is more open than is the case with the related enzymes lipoamide dehydrogenase and glutathione reductase, both of which can be alkylated only on one nascent thiol. Enhanced nucleophilicity of the active site thiols is consistent with the proposed chemical mechanism of thioredoxin reductase. The sequence of the amino-terminal 16 residues is presented.
D000595:Amino Acid Sequence; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D005033:Ethylmaleimide; D006863:Hydrogen-Ion Concentration; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000302:isolation & purification; D013379:Substrate Specificity; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism*
D010446:Peptide Fragments; D009247:NADH, NADPH Oxidoreductases; D013880:Thioredoxin-Disulfide Reductase; D005033:Ethylmaleimide