Centre for Bioinformatics | Pondicherry University
Figure 9. Pre-steady-state kinetics of MtATP-PRT inhibition by -histidine. (A) Representative traces of the MtATP-PRT reaction under pre-steady-state conditions at various -histidine concentrations, and fixed saturating concentrations of ATP, PRPP, and metals. Gray thick lines represent data and black lines the best fits to eq . Data are averages of 10 traces. (B) Plot of the burst amplitude vs -histidine concentration (top) and plot of obs vs -histidine concentration (bottom). Symbols represent data and solid lines best fits to eqs and for burst amplitude and burst rate dependencies, respectively. Reactions were conducted in 50 mM Tris-HCl (pH 8.5), 150 mM KCl, 10 mM MgCl2, 1.5 mM PRPP, 4 mM ATP, and 10 μM MtATP-PRT, at 25 °C. Results are representative of two independent experiments.
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MtATP-phosphoribosyltransferase catalyzes the first and committed step in -histidine biosynthesis in 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 -histidine. Steady-state kinetic studies demonstrate that -histidine inhibition is uncompetitive versus ATP and noncompetitive versus PRPP. At pH values close to neutrality, a ii value of 4 μM was obtained for -histidine. Pre-steady-state kinetic experiments indicate that chemistry is not rate-limiting for the overall reaction and that -histidine inhibition is caused by trapping the enzyme in an inactive conformation. The pH dependence of binding, obtained by nuclear magnetic resonance, indicates that -histidine binds better as the neutral α-amino group. The pH dependence of inhibition (ii), on the contrary, indicates that -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.
To see whether inhibition is caused by nuclear or cytoplasmic events, two groups have recently examined the ability of enucleated cells to autoregulate tubulin synthesis.1,2 These experiments have demonstrated that transcription, processing, and transport of tubulin mRNAs from the nucleus to the cytoplasm are not major sites of autoregulation.