Probing the catalytic roles of n₂‐site glutamate residues in Escherichia coli glutamine synthetase by mutagenesis

Abstract

The contribution of metal ion ligand type and charge to catalysis and regulation at the lower affinity metal ion site (n2 site) of Escherichia coli glutamine synthetase (GS) was tested by mutagenesis and kinetic analysis. The 2 glutamate residues at the n2 site, E129 and E357, were changed to E129D, E129H, E357H, E357Q, and E357D, representing conservative and nonconservative alterations. Unadenylylated and fully adenylylated enzyme forms were studied. The Mn2+-KD values, UV-vis, and fluorescence emission properties were similar for all mutants versus WTGS, except E129H. For kinetic determinations with both MnZ+ and Mg2+, nonconservative mutants (E357H, E129H, E357Q) showed lower biosynthetic activities than conservative mutants (E129D, E357D). Relative to WTGS, all the unadenylylated Mn2+-activated enzymes showed reduced k,,,/K,,, values for ATP (>7-fold) and for glutamate (> 10-fold). Of the unadenylylated Mg2+-activated enzymes, only E129D showed kinetic parameters competitive with WTGS, and adenylylated E129D was a 20-fold better catalyst than WTGS. We propose the n2-site metal ion activates ADP for departure in the phosphorylation of glutamate by ATP to generate y-glutamyl phosphate. Alteration of the charge density at this metal ion alters the transition-state energy for phosphoryl group transfer and may affect ATP binding and/or ADP release. Thus, the steady-state kinetic data suggest that modifying the charge density increases the transition-state energies for chemical steps. Importantly, the data demonstrate that each ligand position has a specialized spatial environment and the charge of the ligand modulates the catalytic steps occurring at the metal ion. The data are discussed in the context of the known X-ray structures of GS.

Structure Prediction and Active Site Analysis of the Metal Binding Determinants in γ-Glutamylcysteine Synthetase

“…The data are consistent with studies in GS where the n2 metal plays a role in ATP binding, as evidenced by equilibrium binding and kinetic studies (32). Mutation of residues analogous to Glu-53 and Glu-489 in GS (Glu-129 and Glu-357) results in enzyme with very low catalytic activity (37). However, mutation of His-269 in GS did not significantly affect ATP binding (38), whereas the Q321A mutant dramatically affected K m app for ATP.…”

“…In contrast, activation of Q321A ␥-GCS by Mn 2ϩ results from the fact that the K m for MnATP is significantly lower than for MgATP. Differential effects on catalytic efficiency between Mn 2ϩ and Mg 2ϩ have also been described for the n2 metal ligand mutants in GS (37,38) and for phosphodiesterase-5 (40). Mn 2ϩ forms bidentate carboxylate complexes with ligands whereas Mg 2ϩ cannot (41).…”

Arginine 302 (Helix IX) in the Lactose Permease of Escherichia coli Is in Close Proximity to Glutamate 269 (Helix VIII) as Well as Glutamate 325 (Helix X)

“…The result also shows that the mutant permease at a concentration of 45 µM causes a 50% reduction in amplitude of the EPR spectrum of 100 µM Mn 2+ , suggesting that the binding stoichiometry may slightly exceed unity. Previous studies show that Glu residues are able to participate in metal chelation (Witmer et al, 1994;He et al, 1995a). Since Glu325 is in close proximity to E269H and R302H, and all three residues are at about the same depth into the membrane, it is possible that Glu325 is responsible for this effect.…”

Nitrogen and Carbon Status Are Integrated at the Transcriptional Level by the Nitrogen Regulator NtrC In Vivo

“…The cellular nitrogen demand (based on 0.3 pg [dry weight]/cell for E. coli and 14% nitrogen content) ( 44 ), and assuming that glutamate is exclusively anabolised via the GS/GOGAT cycle when grown in glucose-replete conditions ( 3 ), is 1.8 × 10 9 glutamine molecules per cell. The NCM3722 strain had 18,691 GS molecules under these conditions ( Table 2 ), and assuming a k cat value of 50 s −1 as determined in vitro ( 45 ), this would be sufficient to support a generation time of 42 min (producing 2.36 × 10 9 glutamine molecules/42 min), whereas the fewer GS molecules in the NCM3722Δ glnG strain (4,456; producing 5.61 × 10 8 glutamine molecules/42 min) presumably would not.…”