Abstract
In the spatial structure of tryptophanase from Proteus vulgaris the guanidinium group of arginine 226 forms a salt bridge with the 3′-oxygen atom of the coenzyme. The replacement of arginine 226 with alanine using site-directed mutagenesis reduced the affinity of the coenzyme for the protein by one order of magnitude compared to the wild-type enzyme. The catalytic activity of the mutant enzyme in the reaction with L-tryptophan was reduced 105-fold compared to the wild-type enzyme. The rates of the reactions with some other substrates decreased 103-104-fold. The mutant enzyme catalyzed exchange of the C-α-proton in complexes with some inhibitors with rates reduced 102-fold compared to the wild-type enzyme. Absorption and circular dichroism spectra of the mutant enzyme and the enzyme–inhibitor complexes demonstrate that the replacement of arginine 226 with alanine does not significantly affect the tautomeric equilibrium of the internal aldimine, but it leads to an alteration of the optimal conformation of the coenzyme–substrate intermediates.
Similar content being viewed by others
REFERENCES
Snell, E. E. (1975) Adv. Enzymol., 42, 287-333.
Newton, W. A., Morino, Y., and Snell, E. E. (1965) J. Biol. Chem., 240, 1211-1218.
Davis, L., and Metzler, D. (1972) Enzymes, 7, 334-338.
Phillips, R. S., Sundararaju, B., and Faleev, N. G. (2000) J. Am. Chem. Soc., 122, 1008-1114.
Isupov, M. N., Antson, A. A., Dodson, G. G., Dodson, E. J., Dementieva, I. S., Zakomirdina, L. N., Wilson, K. S., Dauter, Z., and Harutyunyan, E. H. (1998) J. Mol. Biol., 276, 603-623.
Boyland, E., Manson, D., and Nery, R. (1962) J. Chem. Soc., 2, 606-612.
Savige, W. E., and Fontana, A. (1980) Int. J. Peptide Protein Res., 15, 285-297.
Kamath, A. V., and Yanofsky, C. (1992) J. Biol. Chem., 267, 19978-19985.
Vieira, J., and Messing, J. (1987) Meth. Enzymol., 153, 1-34.
Taylor, J. W., Ott, J., and Eckstein, F. (1985) Nucleic Acids Res., 13, 8764-8785.
Sanger, F., Nicklen, S., and Coulsen, A. R. (1977) Proc. Natl. Acad. Sci. USA, 74, 5463-5467.
Zakomirdina, L. N., Kulikova, V. V., Gogoleva, O. I., Dementieva, I. S., Faleev, N. G., and Demidkina, T. V. (2002) Biochemistry (Moscow), 67, 1189-1196.
Weber, K., and Osborn, M. (1969) J. Biol. Chem., 244, 4406-4412.
Lowry, O. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem., 193, 265-275.
Phillips, R. S., and Gollnick, P. D. (1989) J. Biol. Chem., 264, 10627-10632.
Suelter, C. H., Wang, J., and Snell, E. E. (1976) FEBS Lett., 66, 230-232.
Morino, Y., and Snell, E. E. (1970) Meth. Enzymol., 17A, 439-446.
Cleland, W. W. (1979) Meth. Enzymol., 63, 103-138.
Glasoe, P. V., and Long, F. A. (1960) J. Phys. Chem., 64, 188-194.
Berezin, A. V., and Klesov, A. A. (1976) Apprentice Course of Chemical and Enzyme Kinetics [in Russian], MGU Publishers, Moscow, p. 168.
Peterson, E. A., and Sober, H. A. (1954) J. Amer. Chem. Soc., 76, 169-175.
Morino, Y., and Snell, E. E. (1967) J. Biol. Chem., 242, 5591-5601.
Klotz, I. M., and Hunston, D. L. (1971) Biochemistry, 76, 169-175.
Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. (1997) Nucleic Acids Res., 25, 3389-3402.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Kulikova, V.V., Zakomirdina, L.N., Bazhulina, N.P. et al. Role of Arginine 226 in the Mechanism of Tryptophan Indole-Lyase from Proteus vulgaris . Biochemistry (Moscow) 68, 1181–1188 (2003). https://doi.org/10.1023/B:BIRY.0000009131.78603.8b
Issue Date:
DOI: https://doi.org/10.1023/B:BIRY.0000009131.78603.8b