Thioredoxin and glutaredoxin: small multi-functional redox proteins with active-site disulphide bonds.

Abstract

Thioredoxin and glutaredoxin are small proteins with a redox-active disulphide/dithiol in the active site (Holmgren, 1985). Both exist in Escherichia colias well as in mammalian cells, and were originally isolated as hydrogen donors for ribonucleotide reductase (Holmgren, 1986). This essential enzyme is required in all cells to produce deoxyribonucleotides, which are the precursors for synthesis of DNA de nova Through research on glutaredoxin and thioredoxin for more than 10 and 20 years, respectively, we have gained a considerable amount of information regarding the structures and functions of these proteins. However, the identity of the hydrogen donor for ribonucleotide reductase in vivo in different organisms has not yet been established. An outline of the reactions leading to production of deoxyribonucleotides via either the thioredoxin or glutaredoxin system is shown in Fig. 1. The reducing equivalents are stored in NADPH and are transferred via a series of redox-active disulphides to form deoxyribose. Note that the final acceptors have been assumed to be redox-active disulphides of protein B 1 of ribonucleotide reductase. The function of thioredoxin or glutaredoxin as presented in Fig. 1, is thus to reduce a protein disulphide in the active centre of the enzyme. It has been found that both thioredoxin and glutaredoxin will also catalyse reduction cf other disulphides. For thioredoxin this property has been quite extensively used, and a variety of proteins have been shown to contain susceptible disulphides (Holmgren, 1985). A useful property of the thioredoxin system as a reductant is that the reaction can be followed spectrophotometrically (Holmgren, 1984). By recording the consumption of NADPH at 340 nm, quantitative data on the course of reduction of disulphides in the micromolar range can be obtained. For glutaredoxin the general disulphide reductase activity is expressed in its capacity to be a GSH : disulphide transhydrogenase (Holmgren, 1979). A convenient assay for glutaredoxin is thus to follow the reduction of 2-hydroxyethyldisulphide (HED). Both glutaredoxin from E. coli and calf thymus have this GSH : disulphide transhydrogenase activity. In this respect glutaredoxin shows the same activity as enzymes with GSH : disulphide transhydrogenase (or thiol transferase) activity (Meister & Anderson, 1983 and Mannervik, 1986). The primary structure has been determined for glutaredoxin from E. coli (Hoog et al., 1083) and calf thymus (Klintrot et al., 1984). Both these glutaredoxins have the active centre sequence: Cys-Pro-Tyr-Cys in common. E. coli glutaredoxin consists of 85 amino acid residues, whereas calf thymus glutaredoxin has 101 residues. There is a 31%) overall residue identity in the two proteins. Regarding the relationship between glutaredoxin and GSH : disulphide transhydrogenases, the primary structure of pig liver thiol transferase was recently reported (Gan & Wells, 1987). Pig liver thiol transferase was found to exhibit a strong homology to calf thymus glutaredoxin. However, the active centre sequence in pig thiol transferase was Cys-Pro-Phe-Cys and the peptide chain contained 105 residues. This difference is due to insertion of a tetrapeptide in the structure as compared to calf thymus glutaredoxin. I t is thus not yet clear if glutaredoxin and the cytoplasmic thiol transferase are identical molecules. At present, the evidence favours their being closely related isoenzymes. This would also be in line with the presence of carbohydrate in thiol transferase (Axelsson et al., 1978) which has not been found in glutaredoxin. The thioredoxin system from mammalian cells has certain properties that make it clearly different from the E. coli counterpart, see Table 1. Thus, thioredoxin from calf thymus has two additional structural thiol groups in the C-terminal part of the structure (Engstrom et al., 1,974). Recently, the primary structure of calf thymus thioredoxin was determined (A. Holmgren, C. Palmberg & H. Jornvall, unpublished work). Calf thymus thioredoxin has half-cystiqe residues in positions 31, 34, 61 and 72 in a peptide chain with 104 residues. Upon air oxidation of a fully reduced form of calf thymus thioredoxin, two disulphides are formed both within one molecule. Fully oxidized thioredoxin molecules are enzymically inactive as a substrate for thioredoxin reductase