Rat Liver Glutamine Synthetase: PREPARATION, PROPERTIES, AND MECHANISM OF INHIBITION BY CARBAMYL PHOSPHATE

Abstract

Abstract Glutamine synthetase, purified from rat liver, is homogeneous on acrylamide gel electrophoresis and on ultracentrifugation (s20,w, 15.0 S). The enzyme, which consists of eight subunits (subunit molecular weight, 44,000), resembles ovine brain glutamine synthetase in its physical properties, amino acid composition, and substrate specificity. Complete inhibition of the liver enzyme by methionine sulfoximine is associated with the binding of 4 moles of inhibitor per mole of enzyme. The enzyme is activated by α-ketoglutarate (in the presence of Mg++ or Mn++) and is inhibited by glycine, l-alanine, l-serine, and carbamyl phosphate (with Mn++ only). Evidence is presented that inhibition by carbamyl phosphate is produced by the binding of this compound to the active site of glutamine synthetase. Thus, the enzyme can catalyze the synthesis of ATP from carbamyl phosphate and ADP. When glutamine synthetase is incubated with ADP, carbamyl phosphate, and glutamate, glutamine is formed. Enzyme inactivated by treatment with methionine sulfoximine and ATP did not utilize carbamyl phosphate. Liver glutamine synthetase also catalyzes ATP synthesis from ADP and acetyl phosphate; this reaction is competitively inhibited by glutamate. Glutamine synthetases from ovine brain, rat brain, and Escherichia coli also catalyze ATP synthesis from carbamyl phosphate and ADP.