Kinetic and mutagenic studies of the role of the active site residues Asp-50 and Glu-327 of Escherichia coli glutamine synthetase.

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)