Abstract
Rat kidneyγ-glutamylcysteine synthetase (γGCS) was inactivated by reaction with trinitrobenzene sulfonate (TNBS), and the reaction followed pseudo-first-order kinetics. Inactivation kinetics revealed that only one of the amino acid residues modified by TNBS was essential for-γGCS activity. The addition of 10 mM Mg2+ to the TNBS inactivation reaction resulted in a 16-fold increase in the rate of inactivation. Chromatographic analysis on the tryptic hydrolyzates of trinitrophenylated (TNP) derivatives showed that Lys-38 in theγGCS heavy subunit was significantly modified in the presence of Mg2+. In contrast to small changes in the catalytic properties observed by mutation of Lys-38 to Arg, the mutants K38N and K38E had a marked decrease in enzymatic activity and about twofold increase inK m for glutamate. These results suggest that the positively charged Lys-38 may sbe involved in the binding of glutamate toγGCS.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Chang, L. S., and Chang, C. C. (1993).Biochem. Mol. Biol. Int. 32, 697–703.
Chang, L. S., Kuo, K. W., and Chang, C. C. (1993).Biochim. Biophys. Acta 1202, 216–220.
Chang, L. S., Kuo, K. W., Lin, S. R., and Chang, C. C. (1994).J. Protein Chem. 13, 641–648.
Godwin, A. K., Meister, A., O'Dwyer, P. J., Huang, C. S., Hamilton, T. C., and Anderson, M. E. (1992).Proc. Natl. Acad. Sci. USA 89, 3070–3074.
Huang, C. S., Chang, L. S., Anderson, M. E., and Meister, A. (1993a).J. Biol. Chem. 268, 19675–19680.
Huang, C. S., Anderson, M. E., and Meister, A. (1993b).J. Biol. Chem. 268, 20578–20583.
Lowry, O. H., Rosenberg, N. J., Fan, A. L., and Randall, R. J. (1951).J. Biol. Chem. 193, 265–275.
Meister, A., and Anderson, E. A. (1983).Annu. Rev. Biochem. 52, 711–760.
Richman, P. G., and Meister, A. (1975).J. Biol. Chem. 250, 1422–1426.
Seelig, G. F., and Meister, A. (1985). InMethods in Enzymology (Meister, A., ed.), Academic Press, New York, Vol. 113, pp. 379–390.
Seelig, G. F., Simondsen, R. P., and Meister, A. (1984).J. Biol. Chem. 259, 9345–9349.
Sriram, R., and Ali-Osman, F. (1993).Biochem. Mol. Biol. Int. 30, 1053–1060.
Yao, K. S., Godwin, A. K., Ozols, R. F., Hamilton, T. C., and O'Dwyer, P. J. (1993).Cancer Res. 53, 3662–3666.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Chang, Ls. The functional involvement of Lys-38 in the heavy subunit of rat kidney γ-glutamylcysteine synthetase: Chemical modification and mutagenesis studies. J Protein Chem 15, 321–326 (1996). https://doi.org/10.1007/BF01887121
Received:
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF01887121