Human thioredoxin reductase from HeLa cells: Selective alkylation of selenocysteine in the protein inhibits enzyme activity and reduction with NADPH influences affinity to heparin (selenoproteinyredox active centersytransformed cell linesybromoacetate)

Human thioredoxin reductase (TR) contains selenocysteine (Secys) in a redox center [cysteine (Cys)497,Secys-498] near the C-terminus. The essential role of Secys in TR isolated from HeLa cells was demonstrated by the alkylation studies. Reaction of native NADPH reduced enzyme with bromoacetate at pH 6.5 inhibited enzyme activity 99%. Of the incorporated carboxymethyl (CM) group, 1.1 per subunit, >90% was in CM-Secys-498. Alkylation at pH 8 increased the stoichiometry to 1.6 per subunit with additional modification of the Cys-59, Cys-64 disulfide center. A minor tryptic peptide containing both CM-Cys-497 and CM-Secys-498 was isolated from enzyme alkylated at pH 6.5 or at pH 8. Preparations of TR isolated from HeLa cells grown in a fermentor under high aeration contained selenium-deficient enzyme species that had 50% lower activity. Decreasing oxygen to an optimal level increased cell yield, and fully active TR containing one Se per subunit was present. Reduction of fully active enzyme with tris-(2-carboxyethyl) phosphine converted it from a low to a high heparin affinity form. The tris-(2-carboxyethyl) phosphine-reduced enzyme was oxygen-sensitive and lost selenium and catalytic activity unless maintained under strictly anaerobic conditions. This enzyme could be converted to an oxygeninsensitive species by addition of NADPH, indicating that bound pyridine nucleotide is important for enzyme stability. An induced enzyme conformation in which the essential Secys is shielded from oxidative damage could explain these effects. Thioredoxin reductase (TR) is an NADPH-dependent, FADcontaining disulfide reductase that plays an important role in cell proliferation (1). Unlike the well characterized homologues from yeast and prokaryotes, the larger mammalian enzyme is a selenoprotein that contains a selenocysteine (Secys) residue (2) in the sequence -Cys-Secys-Gly (end) at the C terminus of each subunit (3, 4, 5). Catalysis of electron transfer from NADPH to thioredoxin, which in turn is linked to critical components of cell metabolism such as ribonucleotide reductase (6), AP-1 and NF-kB transcription factors (7–10), vitamin K epoxide reductase (11), thiolperoxidase (12), and plasma glutathione peroxidase (13), illustrates the diversity of processes that depend on this selenium-containing TR. The provision of reduced thioredoxin for two very important cell processes, DNA synthesis and gene transcription, implicates TR as a key enzyme in the control of cell growth. The increased amount of TR in mammalian tumor cells compared with normal tissue (4) suggests a role of this enzyme in malignancy development. Reported catalytic activities of mammalian TRs with disulfide substrates such as homologous thioredoxin, Escherichia coli thioredoxin, or the artificial disulfide 5,59-dithiobis(2nitrobenzoic acid) (DTNB) differ considerably depending on the source of TR and the isolation procedure used. Before the identification of the enzyme as a selenoprotein, inadequate precautions to avoid oxidative damage during isolation may have been a factor. Reported variations in enzyme stability, pI values varying from 4.8 (14) to 5.8 (15) for TR from different sources, and observations that in a single purified preparation two forms with distinct pI values of 5.5 and 5.8 (15) or 5.15 and 5.35 (3) occur are further indications of the heterogeneity of highly purified mammalian TR preparations. More recent studies showing dependency of TR activity in cultured cell lines on availability of selenium in growth media (16, 17) indicated an essential role of selenocysteine in the enzyme. As shown (4), TR isolated from HeLa cells and from human lung adenocarcinoma cells contained two major enzyme species separable by heparin–agarose affinity chromatography. The low-affinity enzyme form had strong reactivity with anti-rat liver TR polyclonal antibodies whereas the high-affinity form was not detected by immunoblot assay. In the present study, two forms of TR were isolated from HeLa cells by heparin affinity chromatography and were characterized. Low catalytic activity of one of these forms was correlated with a low selenium content. Reduction of enzyme activity to 1% or less concomitant with selective alkylation of the Secys-498 residue further indicates the essential role of selenocysteine in catalysis. In this case, reaction of native NADPH reduced enzyme with bromoacetate not only inhibited enzyme activity by 99% but also resulted in incorporation of 1.1 equivalents of alkyl group, of which .90% was present in the carboxymethyl (CM)-derivative of Secys-498 and '5% was present in the CM-derivative of Cys-497. Clearly, the susceptibility of this C-terminal redox center to alkylation at pH 6.5, which is near to the optimum pH range for reduction of a disulfide substrate, points to the accessibility of the reactive ionized selenol group under these conditions and its importance for catalytic activity. MATERIALS AND METHODS Materials. [75Se] selenious acid (30.5 Ciymmol Se) was from the Research Reactor Facility (University of Missouri, Columbia) or from Los Alamos National Laboratory (Los Alamos, NM). Bromo[1-14C]acetic acid (57 mCiymmol) was from American Radiolabeled Chemicals (St. Louis). Heatinactivated fetal bovine serum and DMEM medium were from Irvine Scientific. E. coli recombinant thioredoxin and Tris-(2carboxyethyl)phosphine (TCEP) were from Calbiochem. GluThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. 0027-8424y98y958520-6$0.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: TR, thioredoxin reductase; Cys, cysteine; Secys, selenocysteine; CM, carboxymethyl; TCEP, Tris-(2-carboxyethyl)phosphine; DTNB, 5,59-dithiobis(2-nitrobenzoic acid). *To whom reprint requests should be addressed at: Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 3, Room 108, Bethesda, MD 20892. e-mail: [email protected].