title: Constitutively active glutaminase variants provide insights into the activation mechanism of anthranilate synthase.
abstract: The glutamine amidotransferase (GATase) family comprises enzyme complexes which consist of glutaminase and synthase subunits that catalyze in a concerted reaction the incorporation of nitrogen within various metabolic pathways. An important feature of GATases is the strong stimulation of glutaminase activity by the associated synthase. To understand the mechanism of this tight activity regulation, we probed by site-directed mutagenesis four residues of the glutaminase subunit TrpG from anthranilate synthase that are located between the catalytic Cys-His-Glu triad and the synthase subunit TrpE. In order to minimize structural perturbations induced by the introduced exchanges, the amino acids from TrpG were substituted with the corresponding residues of the closely related glutaminase HisH from imidazole glycerol phosphate synthase. Steady-state kinetic characterization showed that, in contrast to wild-type TrpG, two TrpG variants with single exchanges constitutively hydrolyzed glutamine in the absence of TrpE. A reaction assay performed with hydroxylamine as a stronger nucleophile replacing water and a filter assay with radiolabeled glutamine indicated that the formation of the thioester intermediate is the rate-limiting step of constitutive glutamine hydrolysis. Molecular dynamics simulations with wild-type TrpG and constitutively active TrpG variants suggest that the introduced amino acid exchanges result in a distance reduction between the active site Cys-His pair, which facilitates the deprotonation of the sulfhydryl group of the catalytic cysteine and thus enables its nucleophilic attack onto the carboxamide group of the glutamine side chain. We propose that native TrpG in the anthranilate synthase complex is activated by a similar mechanism.
authors.affiliation: Institute of Biophysics and Physical Biochemistry, University of Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany.
authors.forename: Felix
authors.initials: F
authors.lastname: List
authors.forename: Marco
authors.initials: M
authors.lastname: Bocola
authors.forename: Michaela C
authors.initials: MC
authors.lastname: Haeger
authors.forename: Reinhard
authors.initials: R
authors.lastname: Sterner
mesh_terms: D000878:Anthranilate Synthase / Q000378:metabolism*; D055162:Biocatalysis; D004789:Enzyme Activation; D005972:Glutaminase / Q000378:metabolism*; D056004:Molecular Dynamics Simulation
chemical_list: D005972:Glutaminase; D000878:Anthranilate Synthase