Subunit interaction during catalysis: ammonium ion modulation of catalytic steps in the Escherichia coli glutamine synthetase reaction. Theoretical relationships for oxygen transfer catalyzed by Escherichia coli glutamine synthetase during net adenosine 5'-triphosphate cleavage

Abstract

A new approach for assessing if catalytic cooperativity may occur between subunits was applied to E. coli glutamine synthetase. The extent of oxygen exchange between bound [18O]glutamate and phosphate per molecule of glutamine formed was evaluated at various NH4+ concentrations. This allows calculation of the minimum number of reaction reversals in which bound glutamine is converted to bound glutamate prior to release of glutamine. At 1000 .mu.M NH4+, no detectable reversals occurred, and only 1 glutamate oxygen appeared in product phosphate as required by the reaction mechanism. At 10 .mu.M NH4+, > 15 reversals of bound glutamine formation occurred. Controls showed that under the experimental conditions, free glutamine does not become significantly involved in exchange and the reversal of the oxygen exchange steps appears to be limited to bound glutamine. ATP concentration appears to modulate the exchange of oxygen between glutamate and phosphate only slightly. NH4+ either promotes the dissociation of one of the reaction products or decreases the participation of bound products in the exchange. The NH4+ modulation of the oxygen exchange is consistent with binding of NH4+ at 1 catalytic site promoting catalytic events at an alternate catalytic site but does not eliminate all other explanations.