title: Mechanism of feedback allosteric inhibition of ATP phosphoribosyltransferase.
abstract: MtATP-phosphoribosyltransferase catalyzes the first and committed step in l-histidine biosynthesis in Mycobacterium tuberculosis and is therefore subjected to allosteric feedback regulation. Because of its essentiality, this enzyme is being studied as a potential target for novel anti-infectives. To understand the basis for its regulation, we characterized the allosteric inhibition using gel filtration, steady-state and pre-steady-state kinetics, and the pH dependence of inhibition and binding. Gel filtration experiments indicate that MtATP-phosphoribosyltransferase is a hexamer in solution, in the presence or absence of l-histidine. Steady-state kinetic studies demonstrate that l-histidine inhibition is uncompetitive versus ATP and noncompetitive versus PRPP. At pH values close to neutrality, a K(ii) value of 4 μM was obtained for l-histidine. Pre-steady-state kinetic experiments indicate that chemistry is not rate-limiting for the overall reaction and that l-histidine inhibition is caused by trapping the enzyme in an inactive conformation. The pH dependence of binding, obtained by nuclear magnetic resonance, indicates that l-histidine binds better as the neutral α-amino group. The pH dependence of inhibition (K(ii)), on the contrary, indicates that l-histidine better inhibits MtATP-phosphoribosytransferase with a neutral imidazole and an ionized α-amino group. These results are combined into a model that accounts for the allosteric inhibition of MtATP-phosphoribosyltransferase.
authors.affiliation: Mycobacterial Research Division, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
authors.forename: Sònia
authors.initials: S
authors.lastname: Pedreño
authors.forename: João Pedro
authors.initials: JP
authors.lastname: Pisco
authors.forename: Gérald
authors.initials: G
authors.lastname: Larrouy-Maumus
authors.forename: Geoff
authors.initials: G
authors.lastname: Kelly
authors.forename: Luiz Pedro Sório
authors.initials: LP
authors.lastname: de Carvalho
mesh_terms: D001276:ATP Phosphoribosyltransferase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D025461:Feedback, Physiological / Q000502:physiology*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006639:Histidine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D015394:Molecular Structure; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics
chemical_list: D006639:Histidine; D001276:ATP Phosphoribosyltransferase