Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
권호 표지

Critical Role of the Cysteine 323 Residue in the Catalytic Activity of Human Glutamate Dehydrogenase Isozymes

  • Yang, Seung-Ju (Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Department of Biomedical Laboratory Science, College of Health Science, Yonsei University) ;
  • Cho, Eun Hee (Department of Science Education, College of Education, Chosun University) ;
  • Choi, Myung-Min (Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine) ;
  • Lee, Hyun-Ju (Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine) ;
  • Huh, Jae-Wan (Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine) ;
  • Choi, Soo Young (Department of Genetic Engineering, College of Life Sciences, Hallym University) ;
  • Cho, Sung-Woo (Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine)
  • Received : 2004.09.29
  • Accepted : 2004.11.23
  • Published : 2005.02.28
⟨ Previous Next ⟩

Abstract

The role of residue C323 in catalysis by human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) was examined by substituting Arg, Gly, Leu, Met, or Tyr at C323 by cassette mutagenesis using synthetic human GDH isozyme genes. As a result, the $K_m$ of the enzyme for NADH and ${\alpha}-ketoglutarate$ increased up to 1.6-fold and 1.1-fold, respectively. It seems likely that C323 is not responsible for substrate-binding or coenzyme-binding. The efficiency ($k_{cat}/K_m$) of the mutant enzymes was only 11-14% of that of the wild-type isozymes, mainly due to a decrease in $k_{cat}$ values. There was a linear relationship between incorporation of [$^{14}C$]p-chloromercuribenzoic acid and loss of enzyme activity that extrapolated to a stoichiometry of one mol of [$^{14}C$] incorporated per mol of monomer for wild type hGDHs. No incorporation of [$^{14}C$]p-chloromercuribenzoic acid was observed with the C323 mutants. ADP and GTP had no effect on the binding of p-chloromercuribenzoic acid, suggesting that C323 is not directly involved in allosteric regulation. There were no differences between the two hGDH isozymes in sensitivities to mutagenesis at C323. Our results suggest that C323 plays an important role in catalysis by human GDH isozymes.

Keywords

Acknowledgement

Supported by : Korea Science and Engineering Foundation

References

  1. Abe, T., Ishiguro, S. I., Saito, H., Kiyosawa, M., and Tamai, M. (1992) Partially deficient glutamate dehydrogenase activity and attenuated oscillatory potentials in patient with spinocerebellar degeneration. Invest. Ophthalmol. Vis. Sci. 33, 447-452
  2. Ahn, J.-Y., Choi, S. Y., and Cho, S.-W. (1999) Identification of lysine residue involved in inactivation of brain glutamate dehydrogenase isoproteins by o-phthalaldehyde. Biochimie 81, 1123-1129 https://doi.org/10.1016/S0300-9084(99)00349-1
  3. Anagnou, N. P., Seuanez, H., Modi, W., O'rien, S. J., Papamatheakis, J., et al. (1993) Chromosomal mapping of two members of the human glutamate dehydrogenase (GLUD) gene family to chromosomes 10q22.3-q23 and Xq22-q23. Hum. Hered. 43, 351-356 https://doi.org/10.1159/000154158
  4. Bailey, J., Bell, E. T., and Bell, J. E. (1982) Regulation of bovine glutamate dehydrogenase. J. Biol. Chem. 257, 5579-5583
  5. Baker, P. J., Britton, K. L., Rice, D. W., Rob, A., and Stillman, T. J. (1992) Structural consequences of sequence patterns in the fingerprint region of the nucleotide binding fold implications for nucleotide specificity. J. Mol. Biol. 228, 662-671 https://doi.org/10.1016/0022-2836(92)90848-E
  6. Batra, S. P. and Colman, R. F. (1986) Isolation and identification of cysteinyl peptide labeled by 6-[(4-bromo-2,3-dioxobutyl) thio]-6-deaminoadenosine 5′-diphosphate in the reduced diphosphopyridine nucleotide inhibitory site of glutamate dehydrogenase. Biochemistry 25, 3508-3515 https://doi.org/10.1021/bi00360a005
  7. Bryla, J., Michalik, M., Nelson, J., and Erecinska, M. (1994) Regulation of the glutamate dehydrogenase activity in rat islets of Langerhans and its consequence on insulin release. Metabolism 43, 1187-1195 https://doi.org/10.1016/0026-0495(94)90064-7
  8. Carobbio, S., Ishihara, H., Fernandez-Pascual, S., Bartley, C., Martin-Del-Rio, R., et al. (2004) Insulin secretion profiles are modified by overexpression of glutamate dehydrogenase in pancreatic islets. Diabetologia 47, 266-276 https://doi.org/10.1007/s00125-003-1306-2
  9. Cho, S.-W. and Lee, J. E. (1996) Modification of brain glutamate dehydrogenase isoproteins with pyridoxal 5′-phosphate. Biochimie 78, 817-821 https://doi.org/10.1016/S0300-9084(97)84333-7
  10. Cho, S.-W. and Yoon, H.-Y. (1999) Photoaffinity labeling of brain glutamate dehydrogenase isoproteins with an azido-ADP. J. Biol. Chem. 274, 13948-13953 https://doi.org/10.1074/jbc.274.20.13948
  11. Cho, S.-W., Lee, J., and Choi, S. Y. (1995) Two soluble forms of glutamate dehydrogenase isoproteins from bovine brain. Eur. J. Biochem. 233, 340-346 https://doi.org/10.1111/j.1432-1033.1995.340_1.x
  12. Cho, S.-W., Yoon, H.-Y., Ahn, J.-Y., Choi, S. Y., and Kim, T. U. (1998) Identification of an NAD+ binding site of brain glutamate dehydrogenase isoproteins by photoaffinity labeling. J. Biol. Chem. 273, 31125-31130 https://doi.org/10.1074/jbc.273.47.31125
  13. Cho, S.-W., Cho, E. H., Hwang, S.-H., and Choi, S. Y. (1999) Reactive cysteine residue of bovine brain glutamate dehydrogenase isoproteins. Mol. Cells 9, 91-98
  14. Cho, S.-W., Yoon, H.-Y., Ahn, J.-Y., Lee, E.-Y., and Lee, J. (2001) Cassette mutagenesis of Lysine 130 of human glutamate dehydrogenase: An essential residue in catalysis. Eur. J. Biochem. 268, 3205-3213 https://doi.org/10.1046/j.1432-1327.2001.02209.x
  15. Choi, S. Y., Hong, J. W., Song, M.-S., Jeon, S. G., Bahn, J. H., et al. (1999) Different antigenic reactivities of bovine brain glutamate dehydrogenase isoproteins. J. Neurochem. 72, 2162-2169 https://doi.org/10.1046/j.1471-4159.1999.0722162.x
  16. Colon, A., Plaitakis, A., Perakis, A., Berl, S., and Clarke, D. D. (1986) Purification and characterization of a soluble and a particulate glutamate dehydrogenase from a rat brain. J. Neurochem. 46, 1811-1819
  17. Cosson, M. P., Gros, C., and Talbot, J. C. (1976) Identification of a cysteine residue of glutamate dehydrogenase that binds p-chloromercuribenzoic acid. Biochem. Biophys. Res. Commun. 72, 1304-1310 https://doi.org/10.1016/S0006-291X(76)80157-X
  18. Dutuit, M., Didier-Bazes, M., Vergnes, M., Mutin, M., Conjard, A., et al. (2000) Specific alteration in the expression of glial fibrillary acidic protein, glutamate dehydrogenase, and glutamine synthetase in rats with genetic absence epilepsy. Glia 32, 15-24 https://doi.org/10.1002/1098-1136(200010)32:1<15::AID-GLIA20>3.0.CO;2-#
  19. Fersht, A. (1985) In: Enzyme Structure and Mechanism, pp. 98-120, Freeman, W. H. (ed.), New York
  20. Fisher, H. F. (1985) L-Glutamate dehydrogenase from bovine liver. Methods Enzymol. 113, 16-27 https://doi.org/10.1016/S0076-6879(85)13006-5
  21. Frei, B. and Richter, C. (1988) Mono (ADP-ribosylation) in rat liver mitochondria. Biochemistry 27, 529-535 https://doi.org/10.1021/bi00402a004
  22. Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166, 557-580 https://doi.org/10.1016/S0022-2836(83)80284-8
  23. Herrero-Yraola, A., Bakhit, S. M. A., Franke, P., Weise, C., Schweiger, M., et al. (2001) Regulation of glutamate dehydrogenase by reversible ADP-ribosylation in mitochondria. EMBO J. 20, 2404-2412 https://doi.org/10.1093/emboj/20.10.2404
  24. Hussain, M. H., Zannis, V. I., and Plaitakis, A. (1989) Characterization of glutamate dehydrogenase isoproteins purified from the cerebellum of normal subjects and patients with degenerative neurological disorders and from human neoplastic cell lines. J. Biol. Chem. 264, 20730-20735
  25. Jorcke, D., Ziegler, M., Herrero-Yraola, A., and Schweiger, K. (1998) Enzymic cysteine-specific ADP-ribosylation in bovine liver mitochondria. Biochem. J. 332, 189-193
  26. Julliard, J. and Smith, E. L. (1979) Partial amino acid sequence of the glutamate dehydrogenase of human liver and a revision of the sequence of the bovine enzyme. J. Biol. Chem. 254, 3427-3438
  27. J. Neurochem. v.69 Essential active-site lysine of brain glutamate dehydrogenase isoproteins Kim, S.W.;Lee, J.;Song, M.S.;Choi, S.Y.;Cho, S.W.
  28. Kim, S. W., Lee, J., Song, M. S., Choi, S. Y., and Cho, S.-W. (1997) Essential active-site lysine of brain glutamate dehydrogenase isoproteins. J. Neurochem. 69, 418-422
  29. Lee, E. Y., Yoon, H. Y., Ahn, J.-Y., Choi, S. Y., and Cho, S.-W. (2001) Identification of GTP binding site of human glutamate dehydrogenase using cassette mutagenesis and photoaffinity labeling. J. Biol. Chem. 276, 47930-47936
  30. Lee, K. H., Lee, W.-J., Yang, S.-J., Huh, J.-W., Choi, J., et al. (2004) Inhibitory effects of Cimicifuga heracleifolia on glutamate formation and activities of gutamate dehydrogenase in cultured islets. Mol. Cells 17, 509-514
  31. Lilley, K. S. and Engel, P. C. (1992) The essential active-site lysines of clostridial glutamate dehydrogenase : A study with pyridoxal-5′-phosphate. Eur. J. Biochem. 207, 533-540 https://doi.org/10.1111/j.1432-1033.1992.tb17079.x
  32. Mavrothalassitis, G., Tzimagiorgis, G., Mitsialis, A., Zannis, V. I., Plaitakis, A., et al. (1988) Isolation and characterization of cDNA clones encoding human liver glutamate dehydrogenase:evidence for a small gene family. Proc. Natl. Acad. Sci. USA 85, 3494-3498
  33. Michaelidis, T. M., Tzimagiorgis, G., Moschonas, N. K., and Papamatheakis, J. (1993) The human glutamate dehydrogenase gene family: gene organization and structural characterization. Genomics 16, 150-160 https://doi.org/10.1006/geno.1993.1152
  34. Pandey, A., Sheikh, S., and Katiyar, S. S. (1996) Identification of cystein and lysine residues present at the active site of beef liver glutamate dehydrogenase by o-phthalaldehyde. Biochim. Biophys. Acta 1293, 122-128
  35. Plaitakis, A., Berl, S., and Yahr, M. D. (1984) Neurological disorders associated with deficiency of glutamate dehydrogenase. Ann. Neurol. 15, 144-153 https://doi.org/10.1002/ana.410150206
  36. Plaitakis, A., Flessas, P., Natsiou, A. B., and Shashidharan, P. (1993) Glutamate dehydrogenase deficiency in cerebellar degenerations: clinical, biochemical and molecular genetic aspects. Can. J. Neurol. Sci. Suppl. 3, S109-S116
  37. Plaitakis, A., Metaxari, M., and Shashidharan, P. (2000) Nerve tissue-specific (GLUD2) and housekeeping (GLUD1) human glutamate dehydrogenases are regulated by distinct allosteric mechanisms: implications for biologic function. J. Neurochem. 75, 1862-1869 https://doi.org/10.1046/j.1471-4159.2000.0751862.x
  38. Plaitakis, A., Spanaki, C., Mastorodemos, V., and Zaganas, I. (2003) Study of structure-function relationships in human glutamate dehydrogenases reveals novel molecular mechanisms for the regulation of the nerve tissue-specific (GLUD2) isoenzyme. Neurochem. Int. 43, 401-410 https://doi.org/10.1016/S0197-0186(03)00028-7
  39. Shashidharan, P., Michaelidis, T. M., Robakis, N. K., Kresovali, A., Papamatheakis, J., et al. (1994) Novel human glutamate dehydrogenase expressed in neural and testicular tissues and encoded by an X-linked intronless gene. J. Biol. Chem. 269, 16971-16976
  40. Shashidharan, P., Clarke, D. D., Ahmed, N., Moschonas, N., and Plaitakis, A. (1997) Nerve tissue-specific human glutamate dehydrogenase that is thermolabile and highly regulated by ADP. J. Neurochem. 68, 1804-1811
  41. Smith, T. J., Schmidt, T., Fang, J., Wu, J., Siuzdak, G., et al. (2002) The structure of apo human glutamate dehydrogenase details subunit communication and allostery. J. Mol. Biol. 318, 765-777 https://doi.org/10.1016/S0022-2836(02)00161-4
  42. Stanley, C. A., Lieu, Y. K., Hsu, B. Y. L., Burlina, A. B., Greenberg, C. R., et al. (1998) Hyperinsulinism and hyperammonemia in infants with regulatory mutations of the glutamate dehydrogenase gene. New. Engl. J. Med. 338, 1353-1357
  43. Studier, F. W. and Moffatt, B. A. (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189, 113-130 https://doi.org/10.1016/0022-2836(86)90385-2
  44. Syed, S. E.-H., Hornby, D. P., Brown, P. E., Fitton, J. E., and Engel, P. C. (1994) Site and significance of chemically modifiable cysteine residues in glutamate dehydrogenase of Clostridium symbiosum and the use of protection studies to measure coenzyme binding. Biochem. J. 298, 107-113
  45. Teller, J. K., Smith, R. J., McPherson, M. J., Engel, P. C., and Guest, J. R. (1992) Correlation of intron-exon organization with the three-dimensional structure in glutamate dehydrogenase. Eur. J. Biochem. 206, 151-159 https://doi.org/10.1111/j.1432-1033.1992.tb16912.x
  46. Tzimagiorgis, G. and Moschonas, N. K. (1991) Molecular cloning, structure and expression analysis of a full-length mouse brain glutamate dehydrogenase cDNA. Biochim. Biophys. Acta 1089, 250-253
  47. Valinger, Z., Engel, P. C., and Metzler, D. E. (1993) Is pyridoxal 5′-phosphate an affinity label for phosphate-binding sites in proteins? : the case of bovine glutamate dehydrogenase. Biochem. J. 294, 835-839
  48. Yang, S.-J., Huh, J.-W., Kim, M. J., Lee, W.-J., Kim, T. U., et al. (2003) Regulatory effects of 5′-deoxypyridoxal on glutamate dehydrogenase activity and insulin secretion in pancreatic islets. Biochimie 85, 581-586 https://doi.org/10.1016/S0300-9084(03)00092-0
  49. Yang, S.-J., Huh, J.-W., Hong, H.-N., Kim, T. U., and Cho, S.-W. (2004) Important role of Ser443 in different thermal stability of human glutamate dehydrogenase isozymes. FEBS Lett. 562, 59-64 https://doi.org/10.1016/S0014-5793(04)00183-8
  50. Yoon, H.-Y., Cho, E. H., Kwon, H. Y., Choi, S. Y., and Cho, S.-W. (2002a) Importance of glutamate-279 for the coenzyme binding of human glutamate dehydrogenase. J. Biol. Chem. 277, 41448-41454 https://doi.org/10.1074/jbc.M208208200
  51. Yoon, H.-Y., Lee, E.-Y., and Cho, S.-W. (2002b) Cassette mutagenesis and photoaffinity labeling of adenine binding domain of ADP regulatory site within human glutamate dehydrogenase. Biochemistry 41, 6817-6823 https://doi.org/10.1021/bi0121757
  52. Yoon, H.-Y., Cho, E. H., Yang, S.-J., Lee, H.-Y., Huh, J.-W., et al. (2004) Reactive amino acid residues involved in glutamate-binding of human glutamate dehydrogenase isozymes. Biochimie 86, 261-267 https://doi.org/10.1016/j.biochi.2004.04.005
  53. Yorifuji, T., Muroi, J., Uematsu, A., Hiramatsu, H., and Momoi, T. (1999) Hyperinsulinism-hyperammonemia syndrome caused by mutant glutamate dehydrogenase accompanied by novel enzyme kinetics. Hum. Genet. 104, 476-479 https://doi.org/10.1007/s004390050990