The Influence of Protein Concentration on Oligomer Structure and Catalytic Function of Two Pyruvate Decarboxylases |
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Authors: | Steffen Kutter Michael Spinka Michel H J Koch Stephan König |
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Institution: | (1) Institute for Biochemistry & Biotechnology, Faculty for Life Sciences, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120 Halles (Saale), Germany;(2) European Molecular Biology Laboratory (EMBL c/o DESY), Hamburg Outstation, Germany |
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Abstract: | As a general rule protein concentration typical for structural studies differs considerably from that chosen for kinetic investigations.
Consequently, structure-function relationships are often postulated without appropriate knowledge, whether the functional
behaviour of the enzyme is the same in both protein concentration ranges. To deal with this question, substrate activation
kinetics of two well-characterised yeast pyruvate decarboxylases, from Saccharomyces cerevisiae and from Kluyveromyces lactis, were analysed over the broad protein concentration range 2-2,000 μg/mL. Analytical ultracentrifugation and small-angle X-ray
scattering were used to analyse the enzymes’ oligomer structure in aqueous solution. For the upper part of the concentration
range the determined parameters, like catalytic activity, observed substrate activation rates, sedimentation coefficients
and scattering parameters are independent on enzyme concentration changes. No indication of protein aggregation is detectable.
However, significant changes occur at low enzyme concentration. The catalytically active tetramer dissociates progressively
into dimers with comparable catalytic activity, but with significantly accelerated substrate activation. |
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Keywords: | Substrate activation kinetics X-ray scattering Ultracentrifugation Dimer tetramer equilibrium |
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