TAILIEUCHUNG - Báo cáo Y học: Pyruvate decarboxylase from Kluyveromyces lactis An enzyme with an extraordinary substrate activation behaviour

Pyruvate decarboxylase (EC ) was isolated and purified from the yeast Kluyveromyces lactis. The properties of this enzyme relating to the native oligomeric state, the subunit size, the nucleotide sequence of the coding gene(s), the catalytic activity, and protein fluorescence as well as circular dichroism are very similar to those of the well characterized pyruvate decarboxylase species from yeast. Remarkable differences were found in the substrate activation behaviour of the two pyruvate decarboxylases using three independent methods: steady-state kinetics, stopped-flow measurements, and kinetic dilution experiments. . | Eur. J. Biochem. 269 3256-3263 2002 FEBS 2002 doi Pyruvate decarboxylase from Kluyveromyces lactis An enzyme with an extraordinary substrate activation behaviour Florian Krieger Michael Spinka Ralph Golbik Gerhard Hiibner and Stephan Konig Institut fur Biochemie Fachbereich Biochemie Biotechnologie Martin-Luther-Universitat Halle-Wittenberg Halle Saale Germany Pyruvate decarboxylase EC was isolated and purified from the yeast Kluyveromyces lactis. The properties of this enzyme relating to the native oligomeric state the subunit size the nucleotide sequence of the coding gene s the catalytic activity and protein fluorescence as well as circular dichroism are very similar to those of the well characterized pyruvate decarboxylase species from yeast. Remarkable differences were found in the substrate activation behaviour of the two pyruvate decarboxylases using three independent methods steady-state kinetics stopped-flow measurements and kinetic dilution experiments. The dependence of the observed activation rate constant on the substrate concentration of pyruvate decarboxylase from K. lactis showed a minimum at a pyruvate concentration of mM. According to the mechanism of substrate activation suggested this local minimum occurs due to the big ratio of the dissociation constants for the binding of the first regulatory and the second catalytic substrate molecule. The microscopic rate constants of the substrate activation could be determined by a refined fit procedure. The influence of the artificial activator pyruvamide on the activation of the enzyme was studied. Keywords kinetics thiamine diphosphate microscopic rate constants pyruvamide The cytosolic pyruvate decarboxylase PDC is a key enzyme at the branching point of alcoholic fermentation and respiration in yeast some bacteria and plant seeds. It catalyses the nonoxidative decarboxylation of pyruvate to acetaldehyde and carbon dioxide. Thiamine diphosphate TDP and Mg2 .

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