Active oligomeric states of pyruvate decarboxylase and their functional characterization.
Homomeric pyruvate decarboxylase (E.C 4.1.1.1) from yeast consists of dimers and tetramers under physiological conditions, a K(d) value of 8.1 microM was determined by analytical ultracentrifugation. Dimers and monomers of the enzyme could be populated by equilibrium denaturation using urea as denaturant at defined concentrations and monitored by a combination of optical (fluorescence and circular dichroism) and hydrodynamic methods (analytical ultracentrifugation). Dimers occur after treatment with 0.5 M urea, monomers with 2.0 M urea independent of the protein concentration. The structured monomers are catalytically inactive. At even higher denaturant concentrations (6 M urea) the monomers unfold. The contact sites of two monomers in forming a dimer as the smallest enzymatically active unit are mainly determined by aromatic amino acids. Their interactions have been quantified both by structure-theoretical calculations on the basis of the X-ray crystallography structure, and experimentally by binding of the fluorescent dye bis-ANS. The contact sites of two dimers in tetramer formation, however, are mainly determined by electrostatic interactions. Homomeric pyruvate decarboxylase (PDC) is activated by its substrate pyruvate. There was no difference in the steady-state activity (specific activity) between dimers and tetramers. The activation kinetics of the two oligomeric states, however, revealed differences in the dissociation constant of the regulatory substrate (K(a)) by one order of magnitude. The tetramer formation is related to structural consequences of the interaction transfer in the activation process causing an improved substrate utilization.
Institut für Biochemie/Biotechnologie, Martin-Luther-Universität Halle-Wittenberg, Germany. m2of2@mlucom6.urz.uni-halle.de
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Killenberg-Jabs
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Jabs
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Lilie
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Golbik
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Hübner
D001704:Biopolymers / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D007700:Kinetics; D011487:Protein Conformation; D011767:Pyruvate Decarboxylase / Q000737:chemistry / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D014461:Ultracentrifugation
D001704:Biopolymers; D011767:Pyruvate Decarboxylase