Glutamine Hydrolysis by Imidazole Glycerol Phosphate Synthase Displays Temperature Dependent Allosteric Activation
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) time...
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Frontiers Media S.A.
2018-02-01
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| Series: | Frontiers in Molecular Biosciences |
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| Online Access: | http://journal.frontiersin.org/article/10.3389/fmolb.2018.00004/full |
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| author | George P. Lisi Allen A. Currier J. Patrick Loria J. Patrick Loria |
| author_facet | George P. Lisi Allen A. Currier J. Patrick Loria J. Patrick Loria |
| author_sort | George P. Lisi |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-16T08:34:43Z |
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| institution | Directory Open Access Journal |
| issn | 2296-889X |
| language | English |
| last_indexed | 2024-12-16T08:34:43Z |
| publishDate | 2018-02-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Molecular Biosciences |
| spelling | doaj.art-d2161fa8679f44c18d269bea285125592022-12-21T22:37:48ZengFrontiers Media S.A.Frontiers in Molecular Biosciences2296-889X2018-02-01510.3389/fmolb.2018.00004333077Glutamine Hydrolysis by Imidazole Glycerol Phosphate Synthase Displays Temperature Dependent Allosteric ActivationGeorge P. Lisi0Allen A. Currier1J. Patrick Loria2J. Patrick Loria3Department of Chemistry, Yale University, New Haven, CT, United StatesDepartment of Chemistry, Yale University, New Haven, CT, United StatesDepartment of Chemistry, Yale University, New Haven, CT, United StatesDepartment of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, United StatesThe 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.http://journal.frontiersin.org/article/10.3389/fmolb.2018.00004/fullallosteryenzyme dynamicsrelaxation dispersionthermophileNMR |
| spellingShingle | George P. Lisi Allen A. Currier J. Patrick Loria J. Patrick Loria Glutamine Hydrolysis by Imidazole Glycerol Phosphate Synthase Displays Temperature Dependent Allosteric Activation Frontiers in Molecular Biosciences allostery enzyme dynamics relaxation dispersion thermophile NMR |
| title | Glutamine Hydrolysis by Imidazole Glycerol Phosphate Synthase Displays Temperature Dependent Allosteric Activation |
| title_full | Glutamine Hydrolysis by Imidazole Glycerol Phosphate Synthase Displays Temperature Dependent Allosteric Activation |
| title_fullStr | Glutamine Hydrolysis by Imidazole Glycerol Phosphate Synthase Displays Temperature Dependent Allosteric Activation |
| title_full_unstemmed | Glutamine Hydrolysis by Imidazole Glycerol Phosphate Synthase Displays Temperature Dependent Allosteric Activation |
| title_short | Glutamine Hydrolysis by Imidazole Glycerol Phosphate Synthase Displays Temperature Dependent Allosteric Activation |
| title_sort | glutamine hydrolysis by imidazole glycerol phosphate synthase displays temperature dependent allosteric activation |
| topic | allostery enzyme dynamics relaxation dispersion thermophile NMR |
| url | http://journal.frontiersin.org/article/10.3389/fmolb.2018.00004/full |
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