title: Identification of regions of rabbit muscle pyruvate kinase important for allosteric regulation by phenylalanine, detected by H/D exchange mass spectrometry.
abstract: Mass spectrometry has been used to determine the number of exchangeable backbone amide protons and the associated rate constants that are altered when rabbit muscle pyruvate kinase (rM1-PYK) binds either the allosteric inhibitor (phenylalanine) or a nonallosteric analogue of the inhibitor. Alanine is used as the nonallosteric analogue because it binds competitively with phenylalanine but elicits a negligible allosteric inhibition, i.e., a negligible reduction in the affinity of rM1-PYK for the substrate, phosphoenolpyruvate. This experimental design is expected to distinguish changes in the protein caused by effector binding (i.e., those changes common upon the addition of alanine vs phenylalanine) from changes associated with allosteric regulation (i.e., those elicited by the addition of phenylalanine binding, but not alanine binding). High-quality peptic fragments covering 98% of the protein were identified. Changes in both the number of exchangeable protons per peptide and in the rate constant associated with exchange highlight regions of the protein with allosteric roles. The set of allosterically relevant peptides identified by this technique includes residues previously identified by mutagenesis to have roles in allosteric regulation by phenylalanine.
authors: Department of Biochemistry and Molecular Biology, The University of Kansas Medical Center, MS 3030, 3901 Rainbow Boulevard, Kansas City, Kansas 66160, United States.
authors: Charulata B
authors: CB
authors: Prasannan
authors: Maria T
authors: MT
authors: Villar
authors: Antonio
authors: A
authors: Artigues
authors: Aron W
authors: AW
authors: Fenton
mesh_terms: D000494:Allosteric Regulation; D000495:Allosteric Site; D000818:Animals; D013058:Mass Spectrometry; D008958:Models, Molecular; D009132:Muscles / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010649:Phenylalanine / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011770:Pyruvate Kinase / Q000737:chemistry* / Q000378:metabolism*; D011817:Rabbits
chemical_list: D010455:Peptides; D010649:Phenylalanine; D011770:Pyruvate Kinase