The sequence of amino acid residues around the oxidation-reduction active disulfide in yeast glutathione reductase.

A 14-residue peptide containing the oxidation-reduction active cystine residue from yeast glutathione reductase has been isolated from proteolytic digests of the enzyme in which the free sulfhydryl groups had been reacted with N-ethylmaleimide. The sequence of this disulfide-containing peptide was found to be:(see article). The sequence was highly homologous with the active cystine regions in Escherichia coli and pig heart lipoamide dehydrogenase. The sequences of three of the postulated four thiol-containing regions of the enzyme are also presented, as well as evidence supporting the view that the enzyme is composed of two identical subunits.

The sequence was highly homologous with the active cystine regions in Escherichia coli and pig heart lipoamide dehydrogenase.
The sequences of three of the postulated four thiol-containing regions of the enzyme are also presented, as well as evidence supporting the view that the enzyme is composed of two identical subunits.
Both mechanistically and structurally, lipoamide dehydrogenase and glutathione reductase have proved to be more closely * This work was supported in part by Grant AM 09313 from the National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, United States Public Health Service and in the initial phases by a grant from the Michigan Memorial Phoenix Project. This work was in part aided by United States Public Health Service Grant AM 12734 (Equipment Grant) to the Department of Biological Chemistry. related than either is to thioredoxin reductase (3)(4)(5)(6)(12)(13)(14). Thus, in catalysis, lipoamide dehydrogenase and glutathione reductase cycle between the oxidized state and a spectrally characteristic state in which the enzyme has accepted 2 electrons and these are shared between the FAD and the active center disulfide. This intermediate does not seem to be operative in thioredoxin reductase. In addition this latter enzyme has a lower dimer molecular weight (approximately 70,000) than do the other two (approximately 100,000).
The special reactivity as well as the specificity of the active center disulfides in these enzymes are determined by their environment in the proteins. Crucial components in this environment are the near neighbors of the half-cystines in the primary structure. We have therefore undertaken to compare the sequences in the regions of the active center cystines (6). Peptides have been isolated from Escherichia coli lipoamide dehydrogenase (15, 16) and thioredoxin reductase (17, 18) and from pig heart lipoamide dehydrogenase (19-21) containing both halves of the reactive cystine. A feature common to these peptides is that the half-cystines are close to one another in the primary sequence, being separated by 4 other residues in the lipoamide dehydrogenases and by 2 residues in thioredoxin reductase, thus forming tight loops in the polypeptide chain. The E. coli and pig heart lipoamide dehydrogenase sequences (a prokaryote-eukaryote pair) are identical in 14 of 17 overlapping residues. It is suggested that these proteins have been derived by divergent evolution from a common ancestor (19)(20)(21). The E. coli thioredoxin reductase shows no homology to the lipoamide dehydrogenases.
The sequence of amino acid residues around the active center disulfide has not previously been reported for the third member of this group, glutathione reductase. As with the other two enzymes, this is the only cystine residue in each polypeptide chain. The present paper describes the isolation and sequencing of a peptide containing this disulfide as well as partial sequences of peptides containing three of the thiols.

EXPERIMENTAL PROCEDURES
Materials-Yeast glutathione reductase was obtained from Sigma Company and further purified by chromatography on calcium phosphate as described by Massey and Williams (3 tion indicates a molecular weight of 50,200 per FAD.2 There are 52 arginine plus lysine residues per FAD and 52 peptides are found on maps of trypsin digested enzyme (Fig. 1). One NHZterminal residue, glycine, was found on dansylation of yeast glutathione reductase in urea. These results, in combination with those described below where three of four expected thiols and one unique cystine region are isolated, are consistent with the view that two identical protein chains comprise the native enzyme of moledular weight 100,000. Isolation and Sequence of Active Cystine Region-Yeast glutathione reductase (3.88 pmol) was reacted with a loo-fold excess of N-ethyl[i4C]maleimide over FAD in 0.1 M phosphate, 7.0 M GnHCl, pH 6.0, after anaerobic denaturation for 24 hours. After dialysis, the enzyme was exhaustively digested with pepsin (6% w/w) for 24 hours at 40" in 5% formic acid. The resulting peptides were fractionated on a SE-Sephadex (C-25) column (1.5 x 90 cm) (Fig. 2). was digested with 2y0 w/w TPCK-treated trypsin for 8 hours. The resulting fragments were separated by molecular-sieve chromatography on a Sephadex G-10 column (1.5 X 90 cm) equilibrated with 20 mM ammonium bicarbonate. The flow rate was 13.5 ml per hour. The d&sulfide-containing peak was located by amino acid analysis.
This peptide was performic acid oxidized and the sequence was determined by Edman degradation to be: Digested enzyme (75 nmol) was subjected to descending chromatography with pyridine-butanol-acetic acid-water (10: 15:3: 12) for 18 hours, followed by electrophoresis (3,606 volts for 65 min). The radioautogram was developed for 48 hours after spraying with fluorescamine.
terminal lysine was shown to have the sequence: Lys-Ala-Gly-Lys, by subtractive Edman degradation through two steps and then NHz-terminal determination using dansyl chloride.
Peak D was shown to contain a peptide of the following composition: Val(2.0) ,Met(O.S), and the amino acid lysine. The sequence of the tri-peptide was shown to be Val-Val-Met by subtractive Edman degradation through two steps.
Isolation and Sequences of Three Unique Thiol-containing Regions--After reacting the enzyme with N-ethyl[r4C]maleimide as described above, amino acid analysis indicated 1.9 S-succinylcysteine residues per FAD and 2.9 mol of N-ethyl[r4C]maleimide incorporated per mole of FAD based on radioactivity. This is compared with 4 sulfhydryl residues per FAD demonstrated by amino acid analysis of oxidized enzyme.2 Amino acid analysis of native enzyme, after hydrolysis in the presence of Me2S0, yields 5.9 to 6.2 residues of cysteic acid per FAD, indicating 4 thiol residues in addition to the cystine residue. Radioautograms of N-ethyl[W]maleimide labeled yeast glutathione reductase show four radioactive peptides (Fig. 5). SE-Sephadex elution profiles of pepsin-digested enzyme show four separate peaks of radioactivity (Fig. 2). The isolation of three unique cysteine-containing peptides is summarized in Table I. The thiol peptides were isolated by combination of column and paper chromatography and paper electrophoresis. Both N-ethylmaleimide-labeled enzyme, described above, and iodoacetic acid-labeled enzyme were utilized, as well as both tryptic and peptic digests of labeled enzyme. The sequences are as follows:
The fourth major radioactive fraction (Peak Dz in Fig. 2) was further purified by chromatography on DEAE-cellulose. The radioactivity was found to be contained in a highly cationic peptide with the composition: Lysz , His, Arg, , ASX* , Glx2 ,Gly , Met, Ile ,Tyr , Phe. This peptide corresponds to the cationic peptide on a radioautogram; it contained no S-succinylcysteine and the location of the radioactivity was not determined. glutathione reductase and yeast lipoamide dehydrogenase are known. Chemically this is a relatively conservative change since the side chains of these amino acids are virtually identical in volume.

DISCUSSION
The sequence of amino acid residues around the active site disulfide in yeast glutathione reductase shows a very high degree of homology with the analogous region in pig heart lipoamide dehydrogenase. This represents yet another similarity between these closely related enzymes. There is a growing list of enzyme families in which a high degree of homology has been demonstrated around a common active site residue (31). These include the serine proteases, the cysteine proteases, the carboxypeptidases, the ATP-guanidine phosphotransferases (creatine, arginine, and lombricine kinases), and the aldolases. The pyridine nucleotide-disulfide oxidoreductases can now be added to this list. A parallel can be drawn between this group and the aldolases, where the closely related aldolases A and B show extensive homology but do not show homology with transaldolase. Such is the case with glutathione reductase and lipoamide dehydrogenase where there is no homology with thioredoxin reductase around the active center cystine residue. Fig. 6 compares these structures. The two substitutions in the immediate disulfide region between glutathione reductase and pig heart lipoamide dehydrogenase are conservat'ive both chemically and genetically. It is of interest that the prokaryote Escherichia coli lipoamide dehydrogenase has a valine preceding the first half-cystine, whereas the eukaryotic enzymes have a threonine. The possible genetic significance of this change may become clearer when the sequences of E. coli The lack of homology between lipoamide dehydrogenase and thioredoxin reductase, and the apolar nature of the residues around the active center disulfide in lipoamide dehydrogenase, an enzyme with an apolar substrate, led to the postulation that the disulfide region contained important determinants for interaction with the respective substrates (15,17,19). The very high degree of homology between lipoamide dehydrogenase and glutathione reductase suggests that some modification of that postulation is in order; clearly glutathione and lipoamide are very different molecules. It is possible that the apolar region is important in the binding of lipoamide whereas the determinants for binding the larger glutathione molecule are elsewhere. One would predict that ionic interactions would play a role in the binding of anionic glutathione and the nearby lysine residues may function in this way.
Glutathione reductase (3) and lipoamide dehydrogenase (1, 2) cycle in catalysis between the oxidized state and a 2-electron reduced state. The spectral characteristics of this intermediate make it likely that it is a charge transfer complex in which the donor is a thiolate anion and the acceptor is FAD. Thus the reduced disulfide and the flavin share 2 electrons. It seems reasonable then to suggest that the structure of the disulfide region in these enzymes confers a special reactivity on that disulfide.
The resolved spectrum of FAD when bound to glutathione reductase and lipoamide dehydrogenase indicates that the flavin is bound in a hydrophobic region. It was suggested for lipoamide dehydrogenase (15)  lowing multiple van der Waals contacts. Electron sharing between the flavin and the disulfide demands that they be close. Previous studies have indicated that glutathione reductase is composed of two electrophoretically similar subunits and 2 molecules of FAD (3, 7). The subunit molecular weight, based on amino acid analysis of samples of known FAD content, is 50,600.2 Each subunit contains 6 half-cystines; 2 are present as the active center disulfide and 4 as cysteine residues. Only 1 of the cysteine residues reacts readily with iodoacetate or N-ethylmaleimide in the presence of 7 M GnHCl; two others react partially; the fourth reacts very slowly with phenyl mercuric acetate (3). The data presented here would indicate that the subunits of glutathione reductase are identical or very nearly so. Thus, only a single NHz-terminal residue, glycine, was found; only one disulfidecontaining peptide was isolated; the expected number of peptides were found in maps of tryptic digests based on the number of arginine plus lysine residues per FAD; and finally peptides containing three of the expected four thiols were isolated and shown to have unique sequences. None of the sequences around the thiols of glutathione reductase shows obvious homology with any of the seven sequences of thiol-containing peptides in pig heart lipoamide dehydrogenase (20, 21).
The sequences reported here constitute about 10% of the polypeptide chain. Speculation on the relatedness of glutathione reductase and lipoamide dehydrogenase is not possible with so small a proportion of the total sequence. Preliminary work indicates that the disulfide region is located in a 57-residue peptide isolated from glutathione reductase reacted with cyanogen bromide; its sequence, shown in Fig. 6, is identical with the COOH-terminal portion of the cyanogen bromide peptide. A common location for the active cystine residue in the primary structure of glutathione reductase and lipoamide dehydrogenase would provide strong evidence for any evolutionary relationship between these two enzymes.