Kinetic and Structural Analysis of the Ultrasensitive Behavior of Cyanobacterial ADP- glucose Pyrophosphorylase

D.F. GÓMEZ CASATI, M.A. AON Y A.A. IGLESIAS

BIOCHEMICAL JOURNAL

Biochemical Society

Año: 2000 vol. 350 p. 139 - 147

0264-6021

The kinetic and (supra)molecular properties of the ultrasensitivebehaviour of ADP-glucose pyrophosphorylase (AGPase) fromAnabaena PCC 7120 (a cyanobacterium) were exhaustivelystudied. The response of the enzyme toward the allostericactivator 3-phosphoglycerate (3PGA) occurs with ultrasensitivityas a consequence of the cross-talk with the inhibitor Pi. Molecular`crowding' renders AGPase more sensitive to the interplaybetween the allosteric regulators and, consequently, enhances theultrasensitive response. In crowded media, and when orthophosphateis present, the activation kinetics of the enzyme with3PGA proceed with increased co-operativity and reduced affinitytoward the activator. Under conditions of ultrasensitivity, theenzyme's maximal activation takes place in a narrow range of3PGA concentrations. Moreover, saturation kinetics of theenzyme with respect to its substrates, glucose 1-phosphate andATP, were different at low or high 3PGA levels in crowdedmdiscrimination between low or high levels of the activator, whichincreased the affinity toward the substrates and the maximalactivity reached by the enzyme. Studies of ¯uorescence emissionof tryptophan residues, fourth-derivative spectroscopy and sizeexclusionchromatography indicated that the ultrasensitivebehaviour is correlated with intramolecular conformationalchanges induced in the tertiary structure of the homotetramericenzyme. The results suggest a physiological relevance of theultrasensitive response of AGPase in ŠiŠo, since the enzyme couldbe subtly sensing changes in the levels of allosteric regulators andsubstrates, and thus determining the ¯ux of metabolites towardsynthesis of storage polysaccharides.edia. Only under the latter conditions did AGPase exhibit