Significance of Individual Residues at the Regulatory Site of Yeast Pyruvate Decarboxylase for Allosteric Substrate Activation.
The catalytic activity of the allosteric enzyme pyruvate decarboxylase from yeast is strictly controlled by its own substrate pyruvate via covalent binding at a separate regulatory site. Kinetic studies, chemical modifications, cross-linking, small-angle X-ray scattering, and crystal structure analyses have led to a detailed understanding of the substrate activation mechanism at an atomic level with C221 as the core moiety of the regulatory site. To characterize the individual role of the residues adjacent to C221, we generated variants H92F, H225F, H310F, A287G, S311A, and C221A/C222A. The integrity of the protein structure of the variants was established by small-angle X-ray scattering measurements. The analyses of both steady state and transient kinetic data allowed the identification of the individual roles of the exchanged side chains during allosteric enzyme activation. In each case, the kinetic pattern of activation was modulated but not completely abolished. Despite the crucial role of C221, the covalent binding of pyruvate is not obligate for enzyme activation but is a requirement for a kinetically efficient transition from the inactive to the active state. Moreover, only one of the three histidines guiding the activator molecule to the binding pocket, H310, specifically interacts with C221. H310 stabilizes the thiolate form of C221, ensuring a rapid nucleophilic attack of the thiolate sulfur on C2 of the regulatory pyruvate, thus forming a regulatory dyad. The influence of the other two histidines is less pronounced. Substrate activation is slightly weakened for A287G and significantly retarded for S311A.
Department for Enzymology, Institute of Biochemistry & Biotechnology, Faculty of Biosciences, Martin-Luther-University Halle-Wittenberg , 06120 Halle (Saale), Germany.
Michael
M
Spinka
Department for Enzymology, Institute of Biochemistry & Biotechnology, Faculty of Biosciences, Martin-Luther-University Halle-Wittenberg , 06120 Halle (Saale), Germany.
Sebastian
S
Seiferheld
Department for Enzymology, Institute of Biochemistry & Biotechnology, Faculty of Biosciences, Martin-Luther-University Halle-Wittenberg , 06120 Halle (Saale), Germany.
Philipp
P
Zimmermann
Department for Enzymology, Institute of Biochemistry & Biotechnology, Faculty of Biosciences, Martin-Luther-University Halle-Wittenberg , 06120 Halle (Saale), Germany.
Elena
E
Bergner
Department for Enzymology, Institute of Biochemistry & Biotechnology, Faculty of Biosciences, Martin-Luther-University Halle-Wittenberg , 06120 Halle (Saale), Germany.
Anne-Kathrin
AK
Blume
Department for Enzymology, Institute of Biochemistry & Biotechnology, Faculty of Biosciences, Martin-Luther-University Halle-Wittenberg , 06120 Halle (Saale), Germany.
Angelika
A
Schierhorn
Department for Enzymology, Institute of Biochemistry & Biotechnology, Faculty of Biosciences, Martin-Luther-University Halle-Wittenberg , 06120 Halle (Saale), Germany.
Tom
T
Reichenbach
Department for Enzymology, Institute of Biochemistry & Biotechnology, Faculty of Biosciences, Martin-Luther-University Halle-Wittenberg , 06120 Halle (Saale), Germany.
Robert
R
Pertermann
Department for Enzymology, Institute of Biochemistry & Biotechnology, Faculty of Biosciences, Martin-Luther-University Halle-Wittenberg , 06120 Halle (Saale), Germany.
Christiane
C
Ehrt
Department for Enzymology, Institute of Biochemistry & Biotechnology, Faculty of Biosciences, Martin-Luther-University Halle-Wittenberg , 06120 Halle (Saale), Germany.
Stephan
0000-0003-1476-1751
S
König
D000494:Allosteric Regulation; D004789:Enzyme Activation; D007700:Kinetics; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011767:Pyruvate Decarboxylase / Q000737:chemistry* / Q000378:metabolism*; D019289:Pyruvic Acid / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D019289:Pyruvic Acid; D011767:Pyruvate Decarboxylase