Thermodynamics of active-site ligand binding to Escherichia coli glutamine synthetase

Publisher Website
Google Scholar
Abstract
Active-site ligand interactions with dodecameric glutamine synthetase from Escherichia coli have been studied by calorimetry and fluorometry using the nonhydrolyzable ATP analogue 5''-adenylyl imidodiphosphate (AMP-PNP), L-glutamate, L-Met-(S)-sulfoximine, and the transition-state analogue L-Met-(S)-sulfoximine phosphate. Measurements were made with the unadenylylated enzyme at pH 7.1 in the presence of 100 mM KCl and 1.0 mM MnCl2, under which conditions the two catalytically essential metal ion sites per subunit are occupied and the stoichiometry of active-site ligand binding is equal to 1.0 equiv/subunit. Thermodynamic linkage functions indicate that there is strong synergism between the binding of AMP-PNP and L-Met-(S)-sulfoximine (.delta..DELTA.G'' = -6.4 kJ/mol). In contrast, there is a small antagonistic effect between the binding of AMP-PNP and L-glutamate (.delta..DELTA.G'' = +1.4 kJ/mol). Proton effects were negligible (.ltoreq. 0.2 equiv of H+release or uptake/mol) for the different binding reactions. The binding of AMP-PNP (or ATP) to the enzyme is entropically controlled at 303 K with .DELTA.H = +5.4 kJ/mol and .DELTA.S = +150 J/(K .cntdot. mol). At 303 K, the binding of L-glutamate [.DELTA.H = -22.2 kJ/mol) or L-Met-(S)-sulfoximine (.DELTA.H -45.6 kJ/mol with .DELTA.Cp .simeq. -670 .+-. 420 J/(K .cntdot. mol)] to the AMP-PNP.cntdot.Mn.cntdot.enzyme complex is enthalpically controlled with opposing .DELTA.S values of -29 or -46 J/(K .cntdot. mol), respectively. The overall enthalpy change is negative and the overall entropy change is positive for the simultaneous binding of AMP-PNP and L-glutamate or of AMP-PNP and L-Met-(S)-sulfoximine to the enzyme. For the binding of the transition-state analogue L-Met-(S)-sulfoximine phosphate (which inactivates the enzyme by blocking active sites), both enthalpic and entropic contributions also are favorable at 303 K [.DELTA.G'' .simeq. -109 and .DELTA.H = -54.8 kJ/mol of subunit and .DELTA.S .simeq. +180 J/(K .cntdot. mol)].

This publication has 1 reference indexed in Scilit: