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.
Highlights
Allostery is a ubiquitous biological regulatory mechanism that is essential for cell growth and adaptation
Analytical grade chemicals and antibiotics for protein expression were purchased from AmericanBio (Natick, MA). 3-acetylpyridine adenine dinucleotide (APAD) and glutamate dehydrogenase (GDH) used in kinetic assays were purchased from Santa Cruz Biotechnology (Dallas, TX) and Affymetrix (Santa Clara, CA), respectively, and PRFAR was synthesized as previously described (Lipchock and Loria, 2010)
To examine the temperature dependence of IGPS allostery, the glutamine hydrolysis reaction was monitored in the absence and presence of saturating PRFAR from 303 to 353 K
Summary
Allostery is a ubiquitous biological regulatory mechanism that is essential for cell growth and adaptation. Many recent investigations of allostery have concluded that protein dynamics can be as important as secondary, tertiary, or quaternary structures in directing these biochemical mechanisms (Popovych et al, 2006; Petit et al, 2009; Bu and Callaway, 2011; Motlagh et al, 2012, 2014; Manley et al, 2013; Choi et al, 2015). These and other reports have established dynamic allostery as a widespread biological phenomenon, but one of the facets of these mechanisms that has remained unexplored is the effect of temperature. We sought to exploit the thermostability of this allosteric enzyme to further investigate the relationship between temperature, conformational motions, and its allosteric activation
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