PROBING THE CATALYTIC ROLES OF N(2)-SITE GLUTAMATE RESIDUES IN ESCHERICHIA-COLI GLUTAMINE-SYNTHETASE BY MUTAGENESIS

The contribution of metal ion ligand type and charge to catalysis and regulation at the lower affinity metal ion site (n(2) site) of Escheri chia coli glutamine synthetase (GS) was tested by mutagenesis and kine tic analysis. The 2 glutamate residues at the n(2) 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+-K-D values, UV-vis, and fluorescence emission properties were similar for all mutants vers us WTGS, except E129H. For kinetic determinations with both Mn2+ and M g2+ nonconservative mutants (E357H, E129H, E357Q) showed lower biosynt hetic activities than conservative mutants (E129D, E357D). Relative to WTGS, all the unadenylylated Mn2+-activated enzymes showed reduced k( cat)/K-m values for ATP (>7-fold) and for glutamate (>10-fold). Of the unadenylylated Mg2+-activated enzymes, only E129D showed kinetic para meters competitive with WTGS, and adenylylated E129D was a 20-fold bet ter catalyst than WTGS. We propose the n(2)-site metal ion activates A DP for departure in the phosphorylation of glutamate by ATP to generat e gamma-glutamyl phosphate. Alteration of the charge density at this m etal ion alters the transition-state energy for phosphoryl group trans fer and may affect ATP binding and/or ADP release. Thus, the steady-st ate kinetic data suggest that modifying the charge density increases t he transition-state energies for chemical steps. Importantly, the data demonstrate that each ligand position has a specialized spatial envir onment and the charge of the ligand modulates the catalytic steps occu rring at the metal ion. The data are discussed in the context of the k nown X-ray structures of GS.