title: Altering the allosteric pathway in IGPS suppresses millisecond motions and catalytic activity.
abstract: Imidazole glycerol phosphate synthase (IGPS) is a V-type allosteric enzyme, meaning that its catalytic rate is critically dependent on activation by its allosteric ligand,N'-[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide (PRFAR). The allosteric mechanism of IGPS is reliant on millisecond conformational motions for efficient catalysis. We engineered four mutants of IGPS designed to disrupt millisecond motions and allosteric coupling to identify regions that are critical to IGPS function. Multiple-quantum Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments and NMR chemical shift titrations reveal diminished enzyme flexibility and a reshaping of the allosteric connectivity in each mutant construct, respectively. The functional relevance of the observed motional quenching is confirmed by significant reductions in glutaminase kinetic activity and allosteric ligand binding affinity. This work presents relevant conclusions toward the control of protein allostery and design of unique allosteric sites for potential enzyme inhibitors with regulatory or therapeutic benefit.
authors.affiliation: Department of Chemistry, Yale University, New Haven, CT 06520.
authors.forename: George P
authors.initials: GP
authors.lastname: Lisi
authors.affiliation: Department of Chemistry, Yale University, New Haven, CT 06520.
authors.forename: Kyle W
authors.initials: KW
authors.lastname: East
authors.affiliation: Department of Chemistry, Yale University, New Haven, CT 06520.
authors.forename: Victor S
authors.initials: VS
authors.lastname: Batista
authors.affiliation: Department of Chemistry, Yale University, New Haven, CT 06520; patrick.loria@yale.edu.|Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06520.
authors.forename: J Patrick
authors.initials: JP
authors.lastname: Loria
mesh_terms: D000494:Allosteric Regulation; D000619:Aminohydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D055785:Gene Knockdown Techniques; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D040681:Structural Homology, Protein; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics; D018440:beta-Lactam Resistance
chemical_list: D001426:Bacterial Proteins; C087227:imidazole glycerol phosphate synthase; D000619:Aminohydrolases
keywords: NMR; allostery; community networks; millisecond motions