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Subunit interactions in yeast glyceraldehyde-3-phosphate dehydrogenase.
Metadata
Journalbiochemistry2.865Date
1975-Dec-16
Publication Type
Research Support, U.S. Gov't, P.H.S.
Research Support, U.S. Gov't, Non-P.H.S.
Journal Article
Volume
1975-Dec-16 / 14 : 5428-37
Author
Mockrin SC , Byers LD , Koshland DE
DoiPMIDMESH
Adenosine Triphosphate
Binding Sites
Cyanates
Glyceraldehyde-3-Phosphate Dehydrogenases
Hydrogen-Ion Concentration
Kinetics
Macromolecular Substances
Models, Chemical
NAD
Protein Binding
Protein Conformation
Protein Denaturation
Saccharomyces cerevisiae
Sodium Chloride
Sulfhydryl Compounds
Abstract
The spontaneous inactivation of yeast glyceraldehyde-3-phosphate dehydrogenase was found to fit a simple two-state model at pH 8.5 and 25 degrees. The first step is a relatively rapid dissociation of the tetramer to dimers with the equilibrium largely in favor of the tetramer. In the absence of NAD+ the dimer inactivates irreversibly. The apoenzyme is quite stable with a half-life for complete activity loss proportional to the square root of the enzyme concentration. Perturbances of the protein structure (by pH, ionic strength, and specific salts), which have no effect on the tetrameric state of the molecule, result in an alteration of the cooperativity of NAD+ binding, the reactivity of the active-site sulfhydryl group, and the catalytic activity of the enzyme. Covalent modification of two of the four active-site sulfhydryl groups has profound effects on the enzymic activity which are mediated by changes in the subunit interactions. Sedimentation analysis and hybridization studies indicate that the interaction between subunits remains strong after covalent modification. Under normal physiological and equilibrium dialysis conditions the protein is a tetramer. Equilibrium dialysis studies of NAD+ binding to the enzyme at pH 8.5 and 25 degrees reveal a mixed cooperativity pattern. A model consistent with these observations and the observed half-of-the-sites reactivity is that of ligand induced sequential conformational changes which are transferred across strongly interacting subunit domains. Methods for distinguishing negatively cooperative binding patterns from mixtures of denatured enzyme and multiple species are discussed.
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2.9
Biochemistrybiochemistry
Metadata
LocationUnited States
FromAMER CHEMICAL SOC

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