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. 1997 Jan 1;321(Pt 1):207–210. doi: 10.1042/bj3210207

Identification of an essential cysteine residue in human glutathione synthase.

R R Gali 1, P G Board 1
PMCID: PMC1218055  PMID: 9003420

Abstract

Glutathione is essential for a variety of cellular functions, and is synthesized from gamma-glutamylcysteine and glycine by the action of glutathione synthase (EC 6.3.2.3). Human glutathione synthase is a dimer of two identical subunits, each composed of 474 amino acids. Little is known about the structure-function relationships of mammalian glutathione synthases and, in order to gain a greater understanding of this critical enzyme, we have probed the role of cysteine residues by chemical modification and site-directed mutagenesis. Preincubation with thiol reagents such as p-chloromercuribenzoate, N-ethylmaleimide, iodoacetate and 5,5'-dithiobis-(2-nitrobenzoate) resulted in significant inhibition of recombinant human glutathione synthase. Each subunit contains cysteine residues at positions 294, 409 and 422, and we have prepared four different mutants by replacing individual cysteine residues, or all of the cysteine residues, with alanine. The C294A and C409A cysteine mutants retained significant residual activity, indicating that these two cysteine residues are not essential for activity. In contrast, substantial decreases in enzymic activity were detected with the C422A and cysteine-free mutants. This suggests that Cys-422 may play a significant structural or functional role in human glutathione synthase.

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Selected References

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  1. Board P. G., Pierce K. Expression of human glutathione S-transferase 2 in Escherichia coli. Immunological comparison with the basic glutathione S-transferases isoenzymes from human liver. Biochem J. 1987 Dec 15;248(3):937–941. doi: 10.1042/bj2480937. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Board P. G., Smith J. E., Moore K., Ou D. Erythrocyte gamma-glutamylcysteine synthetase from normal and low-glutathione sheep. Biochim Biophys Acta. 1980 Jun 13;613(2):534–541. doi: 10.1016/0005-2744(80)90109-6. [DOI] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Gali R. R., Board P. G. Sequencing and expression of a cDNA for human glutathione synthetase. Biochem J. 1995 Aug 15;310(Pt 1):353–358. doi: 10.1042/bj3100353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gushima H., Miya T., Murata K., Kimura A. Purification and characterization of glutathione synthetase from Escherichia coli B. J Appl Biochem. 1983 Jun;5(3):210–218. [PubMed] [Google Scholar]
  6. Gushima H., Yasuda S., Soeda E., Yokota M., Kondo M., Kimura A. Complete nucleotide sequence of the E. coli glutathione synthetase gsh-II. Nucleic Acids Res. 1984 Dec 21;12(24):9299–9307. doi: 10.1093/nar/12.24.9299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Habenicht A., Hille S., Knöchel W. Molecular cloning of the large subunit of glutathione synthetase from Xenopus laevis embryos. Biochim Biophys Acta. 1993 Sep 23;1174(3):295–298. doi: 10.1016/0167-4781(93)90202-o. [DOI] [PubMed] [Google Scholar]
  8. Hara T., Tanaka T., Kato H., Nishioka T., Oda J. Site-directed mutagenesis of glutathione synthetase from Escherichia coli B: mapping of the gamma-L-glutamyl-L-cysteine-binding site. Protein Eng. 1995 Jul;8(7):711–716. doi: 10.1093/protein/8.7.711. [DOI] [PubMed] [Google Scholar]
  9. Hibi T., Kato H., Nishioka T., Oda J., Yamaguchi H., Katsube Y., Tanizawa K., Fukui T. Use of adenosine (5')polyphospho(5')pyridoxals to study the substrate-binding region of glutathione synthetase from Escherichia coli B. Biochemistry. 1993 Feb 16;32(6):1548–1554. doi: 10.1021/bi00057a020. [DOI] [PubMed] [Google Scholar]
  10. Huang C. S., He W., Meister A., Anderson M. E. Amino acid sequence of rat kidney glutathione synthetase. Proc Natl Acad Sci U S A. 1995 Feb 14;92(4):1232–1236. doi: 10.1073/pnas.92.4.1232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kato H., Tanaka T., Nishioka T., Kimura A., Oda J. Role of cysteine residues in glutathione synthetase from Escherichia coli B. Chemical modification and oligonucleotide site-directed mutagenesis. J Biol Chem. 1988 Aug 25;263(24):11646–11651. [PubMed] [Google Scholar]
  12. Kato H., Tanaka T., Yamaguchi H., Hara T., Nishioka T., Katsube Y., Oda J. Flexible loop that is novel catalytic machinery in a ligase. Atomic structure and function of the loopless glutathione synthetase. Biochemistry. 1994 May 3;33(17):4995–4999. doi: 10.1021/bi00183a001. [DOI] [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Majerus P. W., Brauner M. J., Smith M. B., Minnich V. Glutathione synthesis in human erythrocytes. II. Purification and properties of the enzymes of glutathione biosynthesis. J Clin Invest. 1971 Aug;50(8):1637–1643. doi: 10.1172/JCI106652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Meister A., Anderson M. E. Glutathione. Annu Rev Biochem. 1983;52:711–760. doi: 10.1146/annurev.bi.52.070183.003431. [DOI] [PubMed] [Google Scholar]
  16. Meister A. Glutathione synthetase from rat kidney. Methods Enzymol. 1985;113:393–399. doi: 10.1016/s0076-6879(85)13052-1. [DOI] [PubMed] [Google Scholar]
  17. Meister A. Glutathione synthetase from rat kidney. Methods Enzymol. 1985;113:393–399. doi: 10.1016/s0076-6879(85)13052-1. [DOI] [PubMed] [Google Scholar]
  18. Mooz E. D., Meister A. Tripeptide (glutathione) synthetase. Purification, properties, and mechanism of action. Biochemistry. 1967 Jun;6(6):1722–1734. doi: 10.1021/bi00858a022. [DOI] [PubMed] [Google Scholar]
  19. Mutoh N., Nakagawa C. W., Ando S., Tanabe K., Hayashi Y. Cloning and sequencing of the gene encoding the large subunit of glutathione synthetase of Schizosaccharomyces pombe. Biochem Biophys Res Commun. 1991 Nov 27;181(1):430–436. doi: 10.1016/s0006-291x(05)81437-8. [DOI] [PubMed] [Google Scholar]
  20. Oppenheimer L., Wellner V. P., Griffith O. W., Meister A. Glutathione synthetase. Purification from rat kidney and mapping of the substrate binding sites. J Biol Chem. 1979 Jun 25;254(12):5184–5190. [PubMed] [Google Scholar]
  21. Peters J. M., Dalrymple B. P., Jorgensen W. K. Sequence of a putative glutathione synthetase II gene and flanking regions from Anaplasma centrale. Biochem Biophys Res Commun. 1992 Feb 14;182(3):1040–1046. doi: 10.1016/0006-291x(92)91836-f. [DOI] [PubMed] [Google Scholar]
  22. Rathbun W. B., Sethna S. S., Van Buskirk G. Purification and properties of glutathione synthetase from bovine lens. Exp Eye Res. 1977 Feb;24(2):145–158. doi: 10.1016/0014-4835(77)90255-x. [DOI] [PubMed] [Google Scholar]
  23. Rawlins M. R., Leaver C. J., May M. J. Characterisation of an Arabidopsis thaliana cDNA encoding glutathione synthetase. FEBS Lett. 1995 Nov 27;376(1-2):81–86. doi: 10.1016/0014-5793(95)01253-1. [DOI] [PubMed] [Google Scholar]
  24. Tanaka T., Kato H., Nishioka T., Oda J. Mutational and proteolytic studies on a flexible loop in glutathione synthetase from Escherichia coli B: the loop and arginine 233 are critical for the catalytic reaction. Biochemistry. 1992 Mar 3;31(8):2259–2265. doi: 10.1021/bi00123a007. [DOI] [PubMed] [Google Scholar]
  25. Yamaguchi H., Kato H., Hata Y., Nishioka T., Kimura A., Oda J., Katsube Y. Three-dimensional structure of the glutathione synthetase from Escherichia coli B at 2.0 A resolution. J Mol Biol. 1993 Feb 20;229(4):1083–1100. doi: 10.1006/jmbi.1993.1106. [DOI] [PubMed] [Google Scholar]

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