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Glutamine Hydrolysis by Imidazole Glycerol Phosphate Synthase Displays Temperature Dependent Allosteric Activation - PubMed

  • ️Mon Jan 01 2018

Glutamine Hydrolysis by Imidazole Glycerol Phosphate Synthase Displays Temperature Dependent Allosteric Activation

George P Lisi et al. Front Mol Biosci. 2018.

Abstract

The enzyme imidazole glycerol phosphate synthase (IGPS) is a model for studies of long-range allosteric regulation in enzymes. Binding of the allosteric effector ligand N'-[5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR) stimulates millisecond (ms) timescale motions in IGPS that enhance its catalytic function. We studied the effect of temperature on these critical conformational motions and the catalytic mechanism of IGPS from the hyperthermophile Thermatoga maritima in an effort to understand temperature-dependent allostery. Enzyme kinetic and NMR dynamics measurements show that apo and PRFAR-activated IGPS respond differently to changes in temperature. Multiple-quantum Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments performed at 303, 323, and 343 K (30, 50, and 70°C) reveal that millisecond flexibility is enhanced to a higher degree in apo IGPS than in the PRFAR-bound enzyme as the sample temperature is raised. We find that the flexibility of the apo enzyme is nearly identical to that of its PRFAR activated state at 343 K, whereas conformational motions are considerably different between these two forms of the enzyme at room temperature. Arrhenius analyses of these flexible sites show a varied range of activation energies that loosely correlate to allosteric communities identified by computational methods and reflect local changes in dynamics that may facilitate conformational sampling of the active conformation. In addition, kinetic assays indicate that allosteric activation by PRFAR decreases to 65-fold at 343 K, compared to 4,200-fold at 303 K, which mirrors the decreased effect of PRFAR on ms motions relative to the unactivated enzyme. These studies indicate that at the growth temperature of T. maritima, PFRAR is a weaker allosteric activator than it is at room temperature and illustrate that the allosteric mechanism of IGPS is temperature dependent.

Keywords: NMR; allostery; enzyme dynamics; relaxation dispersion; thermophile.

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Figures

Figure 1
Figure 1

Structure and reaction of T. maritima IGPS. This HisH subunit, in blue, contains the glutaminase site with the Gln analog acivicin shown in orange sticks. The HisF subunit is colored gray with PRFAR shown in purple sticks. The non-covalent interface of the dimeric complex is highlighted by the dashed black line, which also denotes the subunit responsible for each portion of the chemical reaction.

Figure 2
Figure 2

Analysis of IGPS catalytic activity. Glutaminase profiles in the presence (A) and absence (B) of PRFAR were fit for Michaelis–Menten kinetic parameters and error bars are based on n ≥ 3 measurements and in some cases are smaller than the size of the data point. Traces are color coded as follows: black (303 K), red (313 K), blue (323 K), orange (333 K), green (343 K), and purple (353 K, Basal). Corresponding Arrhenius plots from kinetic traces of activated and basal IGPS are shown in (C) and the temperature dependence of Km is shown in (D). The temperature driven changes in free energy (ΔΔG) between apo and PRFAR-bound IGPS are shown in (E) as a difference of (ΔG)apo – (ΔG)PRFAR obtained using the Eyring expression.

Figure 3
Figure 3

(A) Representative NMR spectral overlays of HisF 13CH3-ILV methyl groups in apo (upper) and PRFAR-bound IGPS (lower) showing temperature-dependent resonance shifts. Resonances in red correspond to spectra collected at 303 K, blue to 323 K, and green to 343 K while arrows indicate the direction of shifts with increasing temperature. Correlations between the temperature dependencies of chemical shifts in 1H13CH3-ILV spectra of apo and PRFAR-bound IGPS are shown for the carbon (B) and proton (C) dimensions. Temperature-dependent shifts outside of 90% confidence boundaries are mapped onto the HisF structure in (D), where areas in black denote residues with proton shifts outside of these boundaries, red denotes carbon shifts outside of these boundaries, and blue denotes residues with both proton and carbon shifts outside of these boundaries.

Figure 4
Figure 4

Representative CPMG curves collected at 600 (left panels) and 800 MHz (right panels) on apo (black) and PRFAR-bound (red) IGPS at (A) 303 K, (B) 323 K, and (C) 343 K. Methyl group assignments are indicated at the top of each column. Error bars were determined from duplicate experiments.

Figure 5
Figure 5

Clustering of kex-values determined from CPMG relaxation dispersion experiments on apo (upper panel) and PRFAR-bound IGPS (lower panel). The distributions of kex-values are shown for experiments carried out at 303, 323, and 343 K according to the inset scales, where optimal bin sizing was determined using a procedure outlined by Scott (1979).

Figure 6
Figure 6

Arrhenius plots for (A) apo and (B) PRFAR-bound IGPS based on temperature-dependent kex-values determined from NMR relaxation dispersion experiments. kex-values were determined from simultaneous fitting of single residue relaxation data obtained at 800 and 600 MHz. A summary of activation energies determined from fitting NMR relaxation data to the Arrhenius equation (data at three temperatures) or the integrated form of the Arrhenius equation (data at two temperatures) is shown in (C) and the resulting values are mapped onto the IGPS structure in (D).

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