Studies on Glutamine Synthetase from Escherichia coli: FORMATION OF PYRROLIDONE CARBOXYLATE AND INHIBITION BY METHIONINE SULFOXIMINE

Abstract

Abstract Both the adenylylated and unadenylylated forms of Escherichia coli glutamine synthetase catalyze the formation of pyrrolidone carboxylate from glutamate in the presence of ATP and divalent metal ions (Mg++ or Mn++) and in the absence of ammonia. The unadenylylated enzyme catalyzes this reaction more rapidly with Mg++ than with Mn++, while the adenylylated enzyme catalyzes it more rapidly with Mn++ than with Mg++. The rates of pyrrolidone carboxylate formation catalyzed by the two forms of the E. coli enzyme are about the same or higher, relative to the corresponding rates of glutamine synthesis, than that catalyzed by ovine brain glutamine synthetase. Both forms of the E. coli enzyme are inhibited when incubated with methionine sulfoximine, ATP, and either Mg++ or Mn++; the unadenylylated enzyme, which is inhibited to about the same extent with Mg++ as with Mn++, is inhibited more rapidly than the adenylylated enzyme. l-Glutamate protects the unadenylylated enzyme (with Mg++ or Mn++) and the adenylylated form (only with Mn++) against inhibition by methionine sulfoximine. Only one of the four stereoisomers of methionine sulfoximine (l-methionine-S-sulfoximine) inhibits the enzyme. Inhibition is associated with tight binding to the enzyme of 9.2 to 11 moles of methionine sulfoximine phosphate per mole of enzyme. E. coli glutamine synthetase acts at a significant rate on several glutamate analogs that are also substrates of the brain enzyme. The data are in accord with the view that the major reaction pathway of glutamine synthesis catalyzed by the E. coli enzyme is similar to that of the reaction catalyzed by the brain enzyme, and thus involves formation and utilization of enzyme-bound γ-glutamyl phosphate and a tetrahedral intermediate or transient state whose structure is analogous to that of l-methionine-S-sulfoximine phosphate.