Covalently Bound Substrate at the Regulatory Site of Yeast Pyruvate Decarboxylases Triggers Allosteric Enzyme Activation

Steffen Kutter,Manfred S Weiss,Georg Wille,Ralph Golbik,Michael Spinka,Stephan König

doi:10.1074/jbc.m806228200

Steffen Kutter, Manfred S Weiss + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m806228200

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Abstract

The mechanism by which the enzyme pyruvate decarboxylase from two yeast species is activated allosterically has been elucidated. A total of seven three-dimensional structures of the enzyme, of enzyme variants, or of enzyme complexes from two yeast species, three of them reported here for the first time, provide detailed atomic resolution snapshots along the activation coordinate. The prime event is the covalent binding of the substrate pyruvate to the side chain of cysteine 221, thus forming a thiohemiketal. This reaction causes the shift of a neighboring amino acid, which eventually leads to the rigidification of two otherwise flexible loops, one of which provides two histidine residues necessary to complete the enzymatically competent active site architecture. The structural data are complemented and supported by kinetic investigations and binding studies, providing a consistent picture of the structural changes occurring upon enzyme activation.

Highlights

The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
Studies on structure function relationships of yeast PDCs showed that the dimer is the minimum functional unit of the enzyme displaying considerable catalytic activity (13, 14)
Kinetic studies reveal a slow isomerization as triggered by substrate binding to a separate regulatory site (15)

Summary

EXPERIMENTAL PROCEDURES

Enzyme Purification—The recombinant wild type and the variants D28A and E477Q have been purified according to the procedures of Killenberg-Jabs et al (3), KlPDC according to Kutter et al (28). Protein Crystallization—KlPDC was dissolved in 20 mM citrate buffer, pH 6.1, 1 mM dithiothreitol, 5 mM ThDP, 5 mM. Chemical structures of the substrate pyruvate, the activators pyruvamide and methyl acetylphosphonate, and the thiohemiketal from pyruvate and cysteine, respectively. The same solution without MAP, but with 7–23% (w/v) PEG 2000/PEG 6000 (1:1 ratio) as precipitant, was used as reservoir. Well diffracting crystals were obtained after 10 days equilibration at 8 °C at 20% PEG and 1 mg of KlPDC per ml.

ScPDC variants were diluted into

Kinetic Effects of MAP

Activator Binding Studies Using SAXS

Structural Implications