A hypothesis on the catalytic mechanism of the selenoenzyme thioredoxin reductase.

Thioredoxin reductase (EC 1.6.4.5), like lipoamide dehydrogenase, mercuric reductase and glutathione reductase, is a member of the pyridine nucleotide-disulphide oxidoreductase family of dimeric flavoenzymes [1]. It catalyses the NADPH-dependent reduction of the 12 kDa protein thioredoxin [NADPH­ H+­thioredoxin-S # ! NADP+­thioredoxin-(SH) # ], which provides reducing equivalents for processes like the reduction of ribonucleotides to deoxyribonucleotides by ribonucleotide reductase [2] and is also considered as a pleiotropic regulator of the cellular redox balance [3,4]. The sequences, sizes and catalytic mechanisms of thioredoxin reductases are different between prokaryotes, such as Escherichia coli, and mammals [5,6]. The human enzyme exhibits an unusually broad substrate specificity. Redox-active proteins, such as thioredoxin, protein disulphide isomerase, natural killer cell lysin (NK-lysin) and calcium-binding proteins 1 and 2, can serve as substrates, as can smaller compounds like selenodiglutathione, S-nitrosoglutathione, lipid hydroperoxides, lipoic acid, -cystine, alloxan, menadione and 5,5«-dithiobis-(2-nitrobenzoic acid) (DTNB) ([2,5,7–9] and references therein). Clearly, any hypothesis for the catalytic mechanism of the enzyme must be able to explain this capability of the protein to reduce so many different substrates. Recently, it has been shown that mammalian thioredoxin reductases are selenoenzymes [6,10–12]. The selenium was identified as a selenocysteine residue, located close to the Cterminus of the protein as the penultimate amino acid [10]. This discovery explains the futile attempts to express active recombinant enzyme in heterologous systems [13]. Mutants lacking the terminal two amino acids do not incorporate the prosthetic group FAD [13]. In a recent study, Arscott et al. [6] have investigated the reductive half-reaction of human placental thioredoxin reductase (hTrxR). Interestingly, not only 2, but almost 3 equivalents of the strong reductant dithionite were required to achieve complete reduction of the enzyme. This observation strongly suggests that, apart from the flavin and the active site thiols, a third redox active site is present in hTrxR. Taking all these data together,we propose theworking hypothesis that the penultimate Cys-Sec pair (Sec is selenocysteine), located on a flexible C-terminal arm, is in redox communication with the active site disulphide}dithiol [6], and might even play an essential role in enzyme catalysis [14]. Demonstration of the concomitant loss of the Cys-Sec pair and enzyme activity would very much substantiate this hypothesis. To study this problem we have chosen the following approach. Human placental thioredoxin reductase (35 units}mg) was purified, essentially as described in [9]. Enzyme activity was determined in the DTNB-reduction assay [5,9]. We then incubated native hTrxR with porcine trypsin in order to remove the last 12 C-terminal amino acids, including the Cys-Sec pair. At different time points, an aliquot was taken, enzyme activitywas determined and the fragments were separated from the protein fraction by ultrafiltration (filter cut-off, 10 kDa). Subsequently, the selenium content in the peptide fraction was determined relative to the enzyme selenium content before trypsin digestion (Figure 1). For selenium determination, two distinct methods, namely atomic absorption spectroscopy and neutron activation analysis, were employed and led to very similar data. The following results were obtained. (1) Only NADPHreduced hTrxR incubated with trypsin showed a significant loss of enzyme activity over time (Figure 1) ; the oxidized form of the enzyme as well as trypsin-free controls maintained their full activity over the time of the experiment. (2) The loss of catalytic activity in the reduced and trypsin-digested sample was directly proportional to the increase of selenium in the peptide fraction (Figure 1). Based on these and the experimental findings cited above, we should like to forward the following hypothesis on the catalytic mechanism of hTrxR. NADPH reduces the active-site disulphide via the flavin. Then the C-terminal redox centre receives the electrons. This is consistent with the results of stopped-flow studies and of static dithionite titrations on the reductive halfreaction of human TrxR [6]. The reduced C-terminal arm then moves to a more exposed position, a view which is supported by the susceptibility of the NADPH reduced enzyme to trypsin digestion, and offers the electrons to the final substrate. That selenium (or the Cys-Sec pair) is essentially involved in catalysis is supported by the fact that loss of selenium leads to a proportional loss in activity. In this context, it should be recalled that disulphide bridges between sequentially adjacent cysteines are strained, due to the comparatively large distance between the unlinked sulphur atoms [15]. Selenium atoms, however, have