NzymeStructure of Human N-Acetyl-L-Glutamate SynthaseFigure 4. NAG binding site. A: Stereo diagram of NAG binding site. The bound NAG is shown in sky-blue sticks. The side-chains involved in Argipressin biological activity hydrogen bonding interactions with NAG are shown in green sticks. The side-chains of other surrounding residues are shown in yellow sticks. The water molecule (w37) is shown in red ball. The electron density map (2Fo c) around bound NAG (contoured at 1.0 s) is shown as blue cage. Potential hydrogen bonding interactions are shown in red dashed lines. B: Stereo diagram of “water wire” 10457188 channel. The bound NAG is shown in sky-blue sticks. Water molecules are shown in yellow balls. Residues involved in hydrogen bonding interactions are shown in brown sticks. Potential hydrogen bonding interactions are shown in red dashed lines. doi:10.1371/journal.pone.0070369.gSite-directed MutagenesisSite-directed mutant DNA sequences encoding hNAT were created using primers containing the desired mutations and the QuikChange Mutagenesis Kit according to the manufacturer’s protocol (Strategene). The sequences of mutant DNA sequences were verified by DNA sequencing.Activity AssayEnzymatic activity was assayed using the method described previously [23]. A stable isotope dilution method using liquid chromatography mass spectrometry (LC S) to measure NAGproduction was adapted. Each assay was Fruquintinib web performed in a 100 ml solution containing 50 mM Tris, pH 8.5, 10 mM glutamate and 2.5 mM AcCoA. The reaction was initiated by the addition of purified recombinant enzyme (20 mg), and the mixture was incubated at 303 K for 5 min and quenched with 100 ml of 30 trichloroacetic acid containing 50 mg of N-acetyl-[13C5]glutamate (13C-NAG) as an internal standard. Precipitated protein was removed by micro-centrifugation. The supernatant (10 ml) was submitted to LC-MS (Agilent) analysis. The mobile phase consisted of 92 solvent A (1 ml trifluoroacetic acid in 1 L water) and 8 solvent B (1 ml trifluoroacetic acid in 1 L of 1:9 water/ acetonitrile) and the flow rate was 0.6 ml/min. Glutamate, NAG,Structure of Human N-Acetyl-L-Glutamate SynthaseFigure 5. Stereo diagram of the proposed CoA binding site. The proposed bound CoA is shown in green sticks. The bound NAG is shown in sky-blue sticks. Side-chains of residues that potentially hydrogen bond to CoA are shown in yellow sticks. The water molecule (w37) that occupies the similar position of thiol S of CoA is shown in a red ball. doi:10.1371/journal.pone.0070369.gand 13C-NAG were detected and quantified by selected ion monitoring mass spectrometry. AcCoA and glutamate titration experiments were carried out with AcCoA or L-glutamate concentration varied in the range of 0.25?.0 and 0.5?0 mM, respectively, and L-glutamate or AcCoA concentration fixed at 10 and 2.5 mM, respectively. The L-glutamate titration data were fit to Michaelis-Menten kinetics, while AcCoA titration data were fit to sigmoidal kinetics (V = Vmax [AcCoA]n/([AcCoA]n+Kmn), where Vmax is maximum activity, Kmis half-maximum activity and n is the Hill coefficient, using the program GNUPLOT.Cross-linking ExperimentCross-linking experiments were performed using the protocol described by Davies and Stark [24]. mNAGS (2.5 mg ) and hNAGS (1.5 and 4.5 mg) were incubated with the cross-linking reagent dimethyl suberimidate (4.5 mg) or suberic acid bis(3-sulfoN-hydroxysuccinimide ester) sodium salt (9.0 mg) in 10 ml solutionFigure 6. Superimposition of hNAT with the NAT domain of subuni.NzymeStructure of Human N-Acetyl-L-Glutamate SynthaseFigure 4. NAG binding site. A: Stereo diagram of NAG binding site. The bound NAG is shown in sky-blue sticks. The side-chains involved in hydrogen bonding interactions with NAG are shown in green sticks. The side-chains of other surrounding residues are shown in yellow sticks. The water molecule (w37) is shown in red ball. The electron density map (2Fo c) around bound NAG (contoured at 1.0 s) is shown as blue cage. Potential hydrogen bonding interactions are shown in red dashed lines. B: Stereo diagram of “water wire” 10457188 channel. The bound NAG is shown in sky-blue sticks. Water molecules are shown in yellow balls. Residues involved in hydrogen bonding interactions are shown in brown sticks. Potential hydrogen bonding interactions are shown in red dashed lines. doi:10.1371/journal.pone.0070369.gSite-directed MutagenesisSite-directed mutant DNA sequences encoding hNAT were created using primers containing the desired mutations and the QuikChange Mutagenesis Kit according to the manufacturer’s protocol (Strategene). The sequences of mutant DNA sequences were verified by DNA sequencing.Activity AssayEnzymatic activity was assayed using the method described previously [23]. A stable isotope dilution method using liquid chromatography mass spectrometry (LC S) to measure NAGproduction was adapted. Each assay was performed in a 100 ml solution containing 50 mM Tris, pH 8.5, 10 mM glutamate and 2.5 mM AcCoA. The reaction was initiated by the addition of purified recombinant enzyme (20 mg), and the mixture was incubated at 303 K for 5 min and quenched with 100 ml of 30 trichloroacetic acid containing 50 mg of N-acetyl-[13C5]glutamate (13C-NAG) as an internal standard. Precipitated protein was removed by micro-centrifugation. The supernatant (10 ml) was submitted to LC-MS (Agilent) analysis. The mobile phase consisted of 92 solvent A (1 ml trifluoroacetic acid in 1 L water) and 8 solvent B (1 ml trifluoroacetic acid in 1 L of 1:9 water/ acetonitrile) and the flow rate was 0.6 ml/min. Glutamate, NAG,Structure of Human N-Acetyl-L-Glutamate SynthaseFigure 5. Stereo diagram of the proposed CoA binding site. The proposed bound CoA is shown in green sticks. The bound NAG is shown in sky-blue sticks. Side-chains of residues that potentially hydrogen bond to CoA are shown in yellow sticks. The water molecule (w37) that occupies the similar position of thiol S of CoA is shown in a red ball. doi:10.1371/journal.pone.0070369.gand 13C-NAG were detected and quantified by selected ion monitoring mass spectrometry. AcCoA and glutamate titration experiments were carried out with AcCoA or L-glutamate concentration varied in the range of 0.25?.0 and 0.5?0 mM, respectively, and L-glutamate or AcCoA concentration fixed at 10 and 2.5 mM, respectively. The L-glutamate titration data were fit to Michaelis-Menten kinetics, while AcCoA titration data were fit to sigmoidal kinetics (V = Vmax [AcCoA]n/([AcCoA]n+Kmn), where Vmax is maximum activity, Kmis half-maximum activity and n is the Hill coefficient, using the program GNUPLOT.Cross-linking ExperimentCross-linking experiments were performed using the protocol described by Davies and Stark [24]. mNAGS (2.5 mg ) and hNAGS (1.5 and 4.5 mg) were incubated with the cross-linking reagent dimethyl suberimidate (4.5 mg) or suberic acid bis(3-sulfoN-hydroxysuccinimide ester) sodium salt (9.0 mg) in 10 ml solutionFigure 6. Superimposition of hNAT with the NAT domain of subuni.