Essential Role of Selenium in the Catalytic Activities of Mammalian Thioredoxin Reductase Revealed by Characterization of Recombinant Enzymes with Selenocysteine Mutations

doi:10.1074/jbc.M000690200

Essential Role of Selenium in the Catalytic Activities of Mammalian Thioredoxin Reductase Revealed by Characterization of Recombinant Enzymes with Selenocysteine Mutations*

Liangwei Zhong and
Arne Holmgren‡

From the Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institute, SE-171 77 Stockholm, Sweden

Abstract

Mammalian thioredoxin reductases (TrxR) are dimers homologous to glutathione reductase with a selenocysteine (SeCys) residue in the conserved C-terminal sequence -Gly-Cys-SeCys-Gly. We removed the selenocysteine insertion sequence in the rat gene, and we changed the SeCys⁴⁹⁸ encoded by TGA to Cys or Ser by mutagenesis. The truncated protein having the C-terminal SeCys-Gly dipeptide deleted, expected in selenium deficiency, was also engineered. All three mutant enzymes were overexpressed in Escherichia coli and purified to homogeneity with 1 mol of FAD per monomeric subunit. Anaerobic titrations with NADPH rapidly generated theA _540 nm absorbance resulting from the thiolate-flavin charge transfer complex characteristic of mammalian TrxR. However, only the SeCys⁴⁹⁸ → Cys enzyme showed catalytic activity in reduction of thioredoxin, with a 100-fold lowerk _cat and a 10-fold lower K _mcompared with the wild type rat enzyme. The pH optimum of the SeCys⁴⁹⁸ → Cys mutant enzyme was 9 as opposed to 7 for the wild type TrxR, strongly suggesting involvement of the low pK _a SeCys selenol in the enzyme mechanism. Whereas H₂O₂ was a substrate for the wild type enzyme, all mutant enzymes lacked hydroperoxidase activity. Thus selenium is required for the catalytic activities of TrxR explaining the essential role of this trace element in cell growth.

Footnotes

↵* This study was supported by the Swedish Cancer Society Project 961, the Swedish Medical Research Council Projects 13X-3529 and 03XS-013005-01A, the K. A. Wallenberg Foundation, and the I.-B. and A. Lundberg Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ To whom correspondence should be addressed. Tel.: 46-8-728-7686; Fax: 46-8-728-4716; E-mail: Arne.Holmgren@mbb.ki.se.
Published, JBC Papers in Press, April 12, 2000, DOI 10.1074/jbc.M000690200
↵2 H. Terashima, GenBank^TM accession number AF053984.
↵3 Zhong, L., Arnér, E. S. J., and Holmgren, A. (2000) Proc. Natl. Acad. Sci. U. S. A., in press.
↵4 L. Zhong, K. Persson, T. Sandalova, G. Schneider, and A. Holmgren, submitted for publication.
↵5 T. Kerimov, S. Kuprin, and A. Holmgren, unpublished results.
Abbreviations:

TrxR

thioredoxin reductase

Trx

thioredoxin

SECIS

selenocysteine insertion sequence

PAGE

polyacrylamide gel electrophoresis

nt

nucleotide

DTNB

5,5′-dithiobis(2-nitrobenzoic acid)

PMSF

phenylmethylsulfonyl fluoride

PCR

polymerase chain reaction
- Received January 31, 2000.
- Revision received April 10, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.