Reaction of both active site thiols of reduced thioredoxin reductase with N-ethylmaleimide

Abstract

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.

Fate Determination of the Flavin Photoreceptions in the Cyanobacterial Blue Light Receptor TePixD (Tll0078)

“…A reduction of oxidized flavin is known to show a bleaching in the 350-500 nm range in the difference spectrum with the isosbestic point at approximately 350 nm. 16 Similar difference spectra were observed during photo-bleaching and dark reversion in the mutant proteins, suggestive of photoreduction of flavin. Further illumination of the mutant proteins for 30 min produced a new broad absorption increase above 600 nm (data not shown), and its spectral properties resemble that of neutral semiquinones.…”

[³H]-13-<i>cis</i>-Retinoic Acid Covalently Binds to Thioredoxin Reductase in Human Keratinocytes

“…The most important feature of this stereospecific suicide inhibition reac tion is the presence of an essential arginine residue in the active site of thioredoxin re ductase. Several years ago, O'Donnell and Williams [26,27] reported on the impor- Fig. 3.…”

Thioredoxin and related proteins in procaryotes

“…Thioredoxin reductase has an M r of 70,000 and consists of two identical subunits, each with a tightly-bound molecule of FAD [95]. Thioredoxin reductase has many properties in common with the other flavoproteins, glutathione reductase, lipoamide dehydrogenase and mercuric reductase [85,[96][97][98]. All facilitate the reduction of disulfide bonds by pyridine nucleotides via the flavin moiety.…”

“…This reactive thiol anion initiates the reaction with the substrate [100][101][102]. In thioredoxin reductase both active site cysteine dithiols will react with N-ethylmaleimide (NEM) at neutral pH [96]. This evidence indicates a more accessible active site in thioredoxin reductase, perhaps due to the fact that, unlike the others, it must reduce another protein.…”