Transfer of Oxygen in the Glutamine Synthetase Reaction

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

“…As shown in Table I, at 1000 µ NH4+ the average number of glutamate oxygens present in P¡ for each P¡ produced, O', was nearly 1.0, in close agreement with that reported by Kowalsky et al (1956). Thus, no apparent reversal of the oxygen-exchange steps occurred.…”

“…Understanding of the experimental design is aided by the schematic representation of the glutamine synthetase reaction with an ADP-trapping system shown in Figure 1. In net glutamine synthesis oxygen is transferred from the -carboxyl group of glutamate to the P¡ formed from ATP (Kowalsky et al, 1956). With glutamine synthetase from E. coli the occurrence of oxygen exchange between enzymebound P¡ and glutamate requires formation of ATP from the P¡.2 Any exchange of oxygen between glutamate and bound P¡ likely occurs prior to the release of ADP since medium ADP is rapidly converted to ATP by the pyruvate kinase trapping system.…”

“…Therefore, the possibility exists that both oxygens originating in a glutamate may appear in product phosphate if extensive reversal of the oxygen-exchange steps occurs prior to ADP release. Kowalsky et al (1956) have shown that when all substrates are present at high concentrations there is a stoichiometric transfer of a single oxygen from glutamate to phosphate during net glutamine synthesis, thus implying there is no reversal of the oxygen-exchange steps. However, if subunit interactions occur such that the binding of a substrate to one subunit influences either a dissociation constant or an oxygen-transfer rate constant on a second subunit, then the potential exists that at low substrate concentrations more than one glutamyl oxygen may be transferred to phosphate during net glutamine synthesis.…”

“…[,80]Glutamate was prepared by equilibrating glutamic acid hydrochloride with highly enriched water in a sealed ampule at 120 °C for 48 h essentially as described by Kowalsky et al (1956). The labeled water was recovered by vacuum transfer and the 180-labeled glutamate was reequilibrated with additional 99% H18OH for another 48 h at 120 °C.…”

“…coli glutamine synthetase appears to lend itself well to this approach for assessment of catalytic cooperativity. The enzyme catalyzes a transfer of an oxygen from the 7-carboxyl group of glutamate to phosphate (Kowalsky et al, 1956;Boyer et al, 1956). Measurement of oxygen exchange involving phosphate or carboxyl groups can be particularly advantageous for detection of catalytic cooperativity since carboxyl or phosphate oxygens of bound substrates may participate equally in catalysis.…”