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Regulation of thiamin diphosphate-dependent 2-oxo acid decarboxylases by substrate and thiamin diphosphate.Mg(II) – evidence for tertiary and quaternary interactions
- Source :
- Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology. 1385:287-306
- Publication Year :
- 1998
- Publisher :
- Elsevier BV, 1998.
-
Abstract
- The regulatory mechanism of substrate activation in yeast pyruvate decarboxylase is triggered by the interaction of pyruvic acid with C221 located on the beta domain at20 A from the thiamin diphosphate (ThDP). To trace the putative information transfer pathway, substitutions were made at H92 on the alpha domain, across the domain divide from C221, at E91, next to H92 and hydrogen bonded to W412, the latter being intimately involved in the coenzyme binding locus. Additional substitutions were made at D28, E51, H114, H115, I415 and E477, all near the active center. The pH-dependent steady-state kinetic parameters, including the Hill coefficient, provide useful insight to this effort. In addition to C221, the residues H92, E91, E51 and H114 and H115 together appear to have a critical impact on the Hill coefficient, providing a pathway for information transfer. To study the activation by ThDP.Mg(II), variants at G231 (of the conserved GDG triplet) and at N258 and C259 (all three being part of the putative ThDP fold) of the E1 component of the Escherichia coli pyruvate dehydrogenase multienzyme complex were studied. Kinetic and spectroscopic evidence suggests that the Mg(II) ligands are very important to activation of the enzymes by cofactors.
- Subjects :
- Models, Molecular
Protein Conformation
Stereochemistry
Biophysics
Pyruvate Dehydrogenase Complex
Crystallography, X-Ray
Biochemistry
Cofactor
Active center
chemistry.chemical_compound
Structural Biology
Pyruvic Acid
Coenzyme binding
Magnesium
Molecular Biology
chemistry.chemical_classification
Binding Sites
biology
Acetyl-CoA
Pyruvate dehydrogenase complex
Protein Structure, Tertiary
Enzyme Activation
Enzyme
chemistry
Mutagenesis, Site-Directed
biology.protein
Pyruvic acid
Thiamine Pyrophosphate
Pyruvate Decarboxylase
Pyruvate decarboxylase
Subjects
Details
- ISSN :
- 01674838
- Volume :
- 1385
- Database :
- OpenAIRE
- Journal :
- Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology
- Accession number :
- edsair.doi.dedup.....077e3c1ff24d560298db94a837bfe3f6