title: Revealing the allosterome: systematic identification of metabolite-protein interactions.
abstract: Small molecule allostery modifies protein function but is not easily discovered. We introduce mass spectrometry integrated with equilibrium dialysis for the discovery of allostery systematically (MIDAS), a method for identifying physiologically relevant, low-affinity metabolite-protein interactions using unmodified proteins and complex mixtures of unmodified metabolites. In a pilot experiment using five proteins, we identified 16 known and 13 novel interactions. The known interactions included substrates, products, intermediates, and allosteric regulators of their protein partners. MIDAS does not depend upon enzymatic measurements, but most of the new interactions affect the enzymatic activity of the protein partner. We found that the fatty acid palmitate interacts with both glucokinase and glycogen phosphorylase. Further characterization revealed that palmitate inhibited both enzymes, possibly providing a mechanism for sparing carbohydrate catabolism when fatty acids are abundant.
authors: Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132, United States.
authors: Thomas
authors: T
authors: Orsak
authors: Tammy L
authors: TL
authors: Smith
authors: Debbie
authors: D
authors: Eckert
authors: Janet E
authors: JE
authors: Lindsley
authors: Chad R
authors: CR
authors: Borges
authors: Jared
authors: J
authors: Rutter
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D000818:Animals; D002417:Cattle; D002853:Chromatography, Liquid; D008401:Gas Chromatography-Mass Spectrometry; D005941:Glucokinase / Q000737:chemistry / Q000378:metabolism; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D024981:Glycogen Phosphorylase / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D055442:Metabolome; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000378:metabolism; D010865:Pilot Projects; D025941:Protein Interaction Mapping; D020543:Proteome / Q000737:chemistry* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology
chemical_list: D020543:Proteome; D005969:Glutamate Dehydrogenase; D024981:Glycogen Phosphorylase; D017853:Phosphotransferases (Alcohol Group Acceptor); D005941:Glucokinase; C021844:NAD kinase