RESUMEN
We have investigated the inhibition of Escherichia coli glutamine synthetase (GS) with alpha- and gamma-substituted analogues of phosphinothricin [L-2-amino-4-(hydroxymethylphosphinyl)butanoic acid (PPT)], a naturally occurring inhibitor of GS. These compounds display inhibition of bacterial GS that is competitive vs L-glutamate, with Ki values in the low micromolar range. At concentrations greater than Ki the phosphinothricins caused time-dependent loss of enzyme activity, while dilution after enzyme inactivation resulted in recovery of enzyme activity. ATP was required for inactivation; the nonhydrolyzable ATP analogue AMP-PCP failed to support inhibition of GS by the phosphinothricins. The binding of these inhibitors to the enzyme was also characterized by measurement of changes in protein fluorescence, which provided similar inactivation rate constants k1 and k2 for the entire series of compounds. Rate constants koff for recovery were also determined by fluorescence measurement and were comparable for both PPT and the gamma-hydroxylated analogue GHPPT and significantly greater for the alpha- and gamma-alkyl-substituted compounds. Electron paramagnetic resonance spectra provided information on the interaction of the phosphinothricins with the manganese form of the enzyme in the absence of ATP, and significant binding was observed for PPT and GHPPT. 31P NMR experiments confirmed that enzyme inactivation is accompanied by hydrolysis of ATP, although phosphorylated phosphinothricins could not be detected in solution. The kinetic behavior of these compounds is consistent with a mechanism involving inhibitor phosphorylation, followed by release from the active site and simultaneous hydrolysis to form Pi and free inhibitor.
Asunto(s)
Aminobutiratos/farmacología , Escherichia coli/enzimología , Glutamato-Amoníaco Ligasa/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Unión Competitiva , Espectroscopía de Resonancia por Spin del Electrón , Activación Enzimática/efectos de los fármacos , Reactivadores Enzimáticos , Glutamato-Amoníaco Ligasa/metabolismo , Cinética , Espectroscopía de Resonancia Magnética , Estructura Molecular , Fosforilación , Espectrometría de Fluorescencia , Relación Estructura-ActividadRESUMEN
The interaction of Escherichia coli glutamine synthetase with beta, gamma-Cr(III)(H2O)4ATP (CrATP) has been studied. This substitution inert nucleotide functioned as an active site directed irreversible inhibitor of glutamine synthetase in solutions containing 15 mM MgCl2, 100 mM KCl, and 10 mM Pipes (pH 6.6). The inactivation reaction followed pseudo-first order saturation kinetics which demonstrated reversible binding of CrATP prior to the formation of inactive enzyme. CrATP was shown to be a competitive inhibitor versus MgATP. Also, significant protection was afforded by MgATP indicating that CrATP inactivates at the active site. Partial protection was afforded by glutamate or inorganic phosphate while inactivation was enhanced by Mn(II). The stoichiometry of CrATP incorporation was approximately one molecule per enzyme subunit, determined spectrophotometrically. Both the delta and lambda isomers of CrATP bound to glutamine synthetase, but only the lambda isomer was an active site directed irreversible inhibitor.
Asunto(s)
Adenosina Trifosfato/análogos & derivados , Marcadores de Afinidad , Cromo , Glutamato-Amoníaco Ligasa/antagonistas & inhibidores , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Sitios de Unión , Unión Competitiva , Escherichia coli/enzimología , Isomerismo , Cinética , MetalesRESUMEN
Chemical modification of a specific arginine residue of Escherichia coli glutamine synthetase has been accomplished by the use of the arginine-specific reagents p-hydroxyphenylglyoxal, phenylglyoxal, and methylglyoxal. Modification of one arginine residue results in complete inactivation of the enzyme and the modified enzyme seems to be extremely stable since no reactivation is observed upon addition of free arginine or dialysis. Saturating levels of ATP but not L-glutamate, L-methionine sulfoximine, or inorganic phosphate provide substantial protection against inactivation of the enzyme suggesting the modified amino acid is at or near the ATP substrate binding site. However, an ATP affinity analog is not prevented from binding upon modification of the arginine residue indicating that the reduction in catalytic activity is not solely due to alteration in substrate binding but may also reflect a catalytic role for the arginine residue.