Abstract
Mutant Arg76Gln and Lys290Gln Saccharomyces cerevisiae phosphoenolpyruvate carboxykinases have been prepared and analyzed. No alteration in the apparent kinetic constants were detected for the Arg76Gln mutant enzyme, while the Lys290Gln mutant showed a 12-fold decrease in V max/K mADP. These results indicate that Arg76 is not involved in CO2 binding, but support the hypothesis that the binding of this substrate induces a conformational change that protects the region around Arg76 from trypsin action [Herrera et al. (1993) J. Protein Chem. 12, 413–418]. These findings also indicate that Lys290, a highly reactive residue against pyrydoxal phosphate [Bazaes et al. (1995), FEBS Lett. 360, 207–210], does not perform an essential function for the enzyme activity.
REFERENCES
Ausabel, M. F., Brent, R., Kington, R. E., Moore, D. D., Smith, J., Siedman, J. G., and Stuhl, K. (1987). In Current Protocols in Molecular Biology, Wiley, New York, Chapter 13.
Bazaes, S., Goldie, H., Cardemil, E., and Jabalquinto, A. M. (1995). FEBS Lett. 360, 207–210.
Cardemil, E., Encinas, M. V., and Jabalquinto, A. M. (1990). Biochim. Biophys. Acta 1040, 71–77.
Chen, C.-Y., Emig, F. A., Schramm, V. L., and Ash, D. E. (1991). J. Biol. Chem. 266, 16645–16652.
Chen, Y. H., Yang, J. T., and Martinez, H. M. (1972). Biochemistry 11, 4120–4131.
Cheng, K.-C., and Nowak, T. (1989). J. Biol. Chem. 264, 19666–19676.
Guidinger, P. F., and Nowak, T. (1991). Biochemistry 30, 8851–8861.
Herrera, L., Encinas, M. V., Jabalquinto, A. M., and Cardemil, E. (1993). J. Protein Chem. 12, 413–418.
Ito, H., Fukuda, Y., Mutata, K., and Kimura, A. (1983). J. Bacteriol. 153, 163–168.
Krautwurst, H., Encinas, M. V., Marcus, F., Latshaw, S. P., Kemp, R. G., Frey, P. A., and Cardemil, E. (1995). Biochemistry 34, 6382–6388.
Kunkel, T. A. (1985). Proc. Natl. Acad. Sci. USA 82, 488–492.
Lins, J., Goldenberg, S., Urbina, J. A., and Anzel, L. M. (1993). Gene 136, 69–77.
Malebrán, L. P., and Cardemil, E. (1987). Biochim. Biophys. Acta 915, 385–392.
Matte, A., Goldie, H., Sweet, R. M., and Delbaere, L. (1996). J. Mol. Biol. 256, 126–143.
Medina, V., Pontarollo, R., Glaeske, D., and Goldie, H. (1990). J. Bacteriol. 172, 7151–7156.
Østerås, M., Finan, T. M., and Stanley, J. (1991). Mol. Gen. Genet. 230, 257–269.
Perrella, F. W. (1988). Anal. Biochem. 174, 437–447.
Rojas, M. C., Encinas, M. V., Kemp, R. G., Latshaw, S. P., and Cardemil, E. (1993). Biochim. Biophys. Acta 1164, 143–151.
Sanger, F., Nicklèn, S., and Coulson, A. A. (1977). Proc. Natl. Acad. Sci. USA 74, 5463–5468.
Stucka, R., Valdes-Hevia, M. D., Gancedo, C., Scharlose, C., and Feldmann, H. (1988). Nucleic Acids Res. 16, 10926.
Tari, L. W., Matte, A., Pugazhenthi, U., Goldie, H., and Delbaere, L. (1996). Nature Struct. Biol. 3, 355–363.
Tortora, P., Hanozet, G. M., and Guerritore, A. (1985). Anal. Biochem. 144, 179–185.
Tsai, M.-D., and Yan, H. (1991). Biochemistry 30, 6806–6818.
Valdes-Hevia, M. D., de la Guerra, R., and Gancedo, C. (1989). FEBS Lett. 258, 313–316.
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Chávez, R., Krautwurst, H. & Cardemil, E. Site-Directed Mutagenesis in Basic Amino Acid Residues of Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase. J Protein Chem 16, 233–236 (1997). https://doi.org/10.1023/A:1026335010370
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DOI: https://doi.org/10.1023/A:1026335010370