title: Conformational changes involving ammonia tunnel formation and allosteric control in GMP synthetase.
abstract: GMP synthetase is the glutamine amidotransferase that catalyzes the final step in the guanylate branch of de novo purine biosynthesis. Conformational changes are required to efficiently couple distal active sites in the protein; however, the nature of these changes has remained elusive. Structural information derived from both limited proteolysis and sedimentation velocity experiments support the hypothesis of nucleotide-induced loop- and domain-closure in the protein. These results were combined with information from sequence conservation and precedents from other glutamine amidotransferases to develop the first structural model of GMPS in a closed, active state. In analyzing this Catalytic model, an interdomain salt bridge was identified residing in the same location as seen in other triad glutamine amidotransferases. Using mutagenesis and kinetic analysis, the salt bridge between H186 and E383 was shown to function as a connection between the two active sites. Mutations at these residues uncoupled the two half-reactions of the enzyme. The chemical events of nucleotide binding initiate a series of conformational changes that culminate in the establishment of a tunnel for ammonia as well as an activated glutaminase catalytic site. The results of this study provide a clearer understanding of the allostery of GMPS, where, for the first time, key substrate binding and interdomain contacts are modeled and analyzed.
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, United States.
authors.forename: Justin C
authors.initials: JC
authors.lastname: Oliver
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, United States.
authors.forename: Ravidra
authors.initials: R
authors.lastname: Gudihal
authors.affiliation: Bindley Bioscience Center, Purdue University, West Lafayette, IN 47907, United States.
authors.forename: John W
authors.initials: JW
authors.lastname: Burgner
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, United States.
authors.forename: Anthony M
authors.initials: AM
authors.lastname: Pedley
authors.affiliation: Department of Pharmaceutical and Administrative Sciences, University of Charleston, Charleston, WV 25304, United States.
authors.forename: Alexander T
authors.initials: AT
authors.lastname: Zwierko
authors.affiliation: Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907, United States.
authors.forename: V Jo
authors.initials: VJ
authors.lastname: Davisson
authors.affiliation: Department of Pharmaceutical and Administrative Sciences, University of Charleston, Charleston, WV 25304, United States. Electronic address: rebeccalinger@ucwv.edu.
authors.forename: Rebecca S
authors.initials: RS
authors.lastname: Linger
mesh_terms: D000494:Allosteric Regulation; D000641:Ammonia / Q000378:metabolism*; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D059748:Proteolysis; D011687:Purines / Q000378:metabolism
chemical_list: D011687:Purines; D000641:Ammonia; D019731:Carbon-Nitrogen Ligases; C020828:GMP synthase (glutamine-hydrolyzing)
keywords: Allosteric control; Ammonia tunnel; Conformational change; Glutamine amidotransferase; Purine biosynthesis; Substrate channeling