Effect of metal ions and adenylylation state on the internal thermodynamics of phosphoryl transfer in the Escherichia coli glutamine synthetase reaction.
Experiments were conducted to study the differences in catalytic behavior of various forms of Escherichia coli glutamine synthetase. The enzyme catalyzes the ATP-dependent formation of glutamine from glutamate and ammonia via a gamma-glutamyl phosphate intermediate. The physiologically important metal ion for catalysis is Mg2+; however, Mn2+ supports in vitro activity, though at a reduced level. Additionally, the enzyme is regulated by a covalent adenylylation modification, and the metal ion specificity of the reaction depends on the adenylylation state of the enzyme. The kinetic investigations reported herein demonstrate differences in binding and catalytic behavior of the various forms of glutamine synthetase. Rapid quench kinetic experiments on the unadenylylated enzyme with either Mg2+ or Mn2+ as the activating metal revealed that product release is the rate-limiting step. However, in the case of the adenylylated enzyme, phosphoryl transfer is the rate-limiting step. The internal equilibrium constant for phosphoryl transfer is 2 and 5 for the unadenylylated enzyme with Mg2+ or Mn2+, respectively. For the Mn2(+)-activated adenylylated enzyme the internal equilibrium constant is 0.1, indicating that phosphoryl transfer is less energetically favorable for this form of the enzyme. The factors that make the unadenylylated enzyme most active with Mg2+ are discussed.
Abell
L M
LM
Department of Chemistry, Pennsylvania State University, University Park 16802.
Villafranca
J J
JJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005974:Glutamate-Ammonia Ligase / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D013816:Thermodynamics
D008345:Manganese; D000255:Adenosine Triphosphate; D005974:Glutamate-Ammonia Ligase; D008274:Magnesium