A METABOLIC CONTROL ANALYSIS OF THE GLUTAMINE-SYNTHETASE GLUTAMATE SYNTHASE CYCLE IN ISOLATED BARLEY (HORDEUM-VULGARE L) CHLOROPLASTS

Ammonia assimilation in chloroplasts occurs via the glutamine syntheta se/glutamate synthase (GS/GOGAT) cycle. To determine the extent to whi ch these enzymes contribute to the control of ammonia assimilation, a metabolic control analysis was performed on isolated barley (Hordeum v ulgare L.) leaf chloroplasts. Pathway flux was measured polarographica lly as onium-plus-2-oxoglutarate-plus-glutamine-dependent O-2 evolutio n in illuminated chloroplasts. Enzyme activity was modulated by titrat ion with specific, irreversible inhibitors of CS (phosphinothricin) an d GOGAT (azaserine). Flux control coefficients (C-E0(j0),) were determ ined (a) by differentiation of best-fit hyperbolic curves of the data sets (flux versus enzyme activity), and (b) from estimates of the devi ation indices (D-El(ji)). Both analyses gave similar values for the co efficients. The control coefficient for CS was relatively high and the value did not change significantly with changes in 9-oxoglutarate con centration (C-E0(j0) = 0.58 at 5 mM 2-oxoglutarate and 0.40 at 20 mM P -oxoglutarate). The control coefficient for GOGAT decreased with decre asing glutamine concentrations, from 0.76 at 20 mM glutamine to 0.19 a t 10 mM glutamine. Thus, at high concentrations of glutamine, GOGAT ex erts a major control over flux with a significant contribution also fr om GS. At lower concentrations of glutamine, however, GOGAT exerts far less control over pathway flux.