title: The influence of protein concentration on oligomer structure and catalytic function of two pyruvate decarboxylases.
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 microg/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.
authors.affiliation: Institute for Biochemistry & Biotechnology, Faculty for Life Sciences, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Str. 3, 06120, Halles (Saale), Germany.
authors.forename: Steffen
authors.initials: S
authors.lastname: Kutter
authors.forename: Michael
authors.initials: M
authors.lastname: Spinka
authors.forename: Michel H J
authors.initials: MH
authors.lastname: Koch
authors.forename: Stephan
authors.initials: S
authors.lastname: König
mesh_terms: D002384:Catalysis; D020134:Catalytic Domain; D019281:Dimerization; D004789:Enzyme Activation; D007700:Kinetics; D007716:Kluyveromyces / Q000201:enzymology*; D011767:Pyruvate Decarboxylase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019289:Pyruvic Acid / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D014461:Ultracentrifugation