The influence of several amino acid residues at the regulatory site of yeast pyruvate decarboxylase on the allosteric substrate activation of this enzyme

Pyruvate decarboxylase (EC 4.1.1.1) is an allosteric enzyme whose catalytic activity is strictly controlled by its substrate pyruvate binding at a separate regulatory site. Chemical enzyme modification and cross-linking, kinetic characterization of several PDC variants, and the determination of their crystal structures in complex with substrates or artificial activators led to a comprehensive understanding of the mechanism of allosteric activation at an atomic level [1-5, 8, 9]. Particularly, the signal transfer pathway from the regulatory to the active site as triggered by activator binding at the regulatory site and resulting in rearrangements of several amino acid residues at the catalytic site could be traced [6, 7]. To understand the role of individual amino acid residues for substrate activation we generated a number of variants with single amino acid substitutions at the regulatory site, e.g. C221A/C222A, H92F, H225F, H310F, A287G, and S311A. The first of the two adjacent cysteine residues, C221, was identified as the core of the activator binding site. Three surrounding histidine residues create a positively charged trap that guides the activator molecule to the binding pocket at C221. Together with the histidine residues, A287 and S311 are able to interact with the activator molecule bound covalently at C221. Structure and function of the variants have been analysed applying detailed steady state and transient kinetic approaches as well as small-angle X-ray solution scattering measurements. The structural integrity of the variants was not influenced by the site specific mutations [10]. While A287G shows a wild type-like behaviour all other variants clearly deviate in their kinetic properties from the wild type. The results obtained allow a deeper look at the details of substrate activation.References1 Hübner, G., König, S. and Schellenberger, A. (1988) Biomed. Biochim. Acta 47, 9-18.2 Lu, G., Dobritzsch, D., Baumann, S., Schneider, G. and König, S. (2000) Eur. J. Biochem. 267, 861-868.3 Killenberg-Jabs, M., König, S., Eberhardt, I., Hohmann, S. and Hübner, G. (1997) Biochemistry 36, 1900-1905.4 Dobritzsch, D., König, S., Schneider, G. and Lu, G. (1998) J. Biol. Chem. 273, 20196-20204.5 Krieger, F., Spinka, M., Golbik, R., Hübner, G. and König, S. (2002) Eur. J. Biochem. 269, 3256-3263.6 Kutter, S., Weiss, M. S., Wille, G., Golbik, R., Spinka, M. and König, S. (2009) J. Biol. Chem. 284, 12136-12144.7 König, S., Spinka, M. and Kutter, S. (2009) J. Mol. Catal. B Enz. 61, 100-110.8 Baburina, I., Gao, Y., Hu, Z., Jordan, F., Hohmann, S. and Furey, W. (1994) Biochemistry 33, 5630-5635.9 Sergienko, E. A. and Jordan, F. (2001) Biochemistry 40, 7355-7368, 7369-7381, 7382-7403.10 König, S, Svergun, D. I., Volkov, V. V., Feigin, L. A. and Koch, M. H. J. (1998) Biochemistry 37, 5329-5334