title: Kinetic and mutagenic studies of the role of the active site residues Asp-50 and Glu-327 of Escherichia coli glutamine synthetase.
abstract: The role of Asp-50 and Glu-327 of Escherichia coli glutamine synthetase in catalysis and substrate binding has been interrogated by construction of site-directed mutants at these positions. Steady-state and rapid-quench kinetic methods were used to elucidate contributions of Asp-50 and Glu-327 to the Km values of all three substrates, ATP, glutamate, and NH4+, as well as to the enzymatic kcat value. Kinetic constants were obtained for the D50A enzyme using both Mg2+ and Mn2+ as activating metal ions; the data reveal that Asp-50 has a significant role in both substrate binding and catalysis as reflected by the increases in the Km values for NH4+ and the destabilization of both the ground state and the transition state for phosphoryl transfer. The D50E mutant was found to have activity with Mn2+ but very low activity with Mg2+, the physiologically important metal ion. The kcat/Km values for all three substrates were substantially altered by changing Asp to Glu. The steady-state results for the E327A mutant indicate a decreased kcat/Km value for NH4+ compared to that of the wild-type enzyme. The E327A-Mg2+ enzyme destabilizes the ground state of the ternary complex (E-ATP-Glu-NH4+) and the transition state for phosphoryl transfer while the E327A-Mn2(+)-enzyme provides greater stabilization for the ATP and glutamate complexes but destabilizes phosphoryl transfer steps in the ternary complex. Overall, these results suggest that Asp-50 is likely involved in binding NH4+ and may also play a role in catalyzing deprotonation of NH4+ to form NH3.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemistry, Pennsylvania State University, University Park 16802.
authors.forename: M
authors.initials: M
authors.lastname: Alibhai
authors.forename: J J
authors.initials: JJ
authors.lastname: Villafranca
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D005974:Glutamate-Ammonia Ligase / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D005971:Glutamates / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D000644:Quaternary Ammonium Compounds / Q000378:metabolism
chemical_list: D005971:Glutamates; D000644:Quaternary Ammonium Compounds; D001224:Aspartic Acid; D018698:Glutamic Acid; D008345:Manganese; D000255:Adenosine Triphosphate; D005974:Glutamate-Ammonia Ligase