Effect of temperature on the activity and stability of ADPglucose pyrophosphorylase

D.F. GÓMEZ CASATI, , J. PREISS, A.A. IGLESIAS

Vancouver, British Columbia, Canada

Congreso; Annual Meeting of the American Society of Plant Biologists (ASPB); 1997

ASPB

ADPglucose pyrophosphorylase is the key regulatory enzyme in metabolic pathways for bacterial glycogen and plant starch synthesis. Purification of the enzyme from different sources generally involves a heat treatment step (5 min at 60 Celsius degrees) carried out in the presence of 5-30 mM Pi. Use of this purification step is empirically performed on the basis of the stability of the pyrophosphorylase under the stated conditions. In the present work we study the effect of temperature on the activity and the stability of ADPglucose pyrophosphorylase purified from cyanobacteria. Optima temperature for enzyme activity of 40 or 50 Celsius degrees were observed, when assays were performed in the absence or in the presence of allosteric effectors (3P-glycerate and Pi), respectively. Arrhenius plots of the pyrophosphorolytic activity of the enzyme as a function of temperature, exhibited no linearity, depending on the conditions of the assay. Incubation of purified ADPglucose pyrophosphorylase at pH 7.5 and temperatures above 45 Celsius degrees resulted in an inactivation of the enzyme which followed first-order kinetics. The presence of ATP, PPi or ADPglucose in the incubation media protected (only partially) the enzyme against thermal inactivation, whereas glucose-1P showed no effect. Effective protections were observed when 3P-glycerate or Pi were present in the incubation media, with the increase of thermal stability of the enzyme being dependent of effector concentration. From protection afforded by different concentrations of effector, dissociation constants of 0.08 and 0.09 mM, respectively, for the interaction of 3P-glycerate and Pi with the enzyme were determined. Other compounds increasing thermal stability of ADPglucose pyrophosphorylase were KCl (1 M), sucrose (64%), polyethyleneglycol (56%) or polyvinylpyrrolidone (34%). Results are discussed in terms of possible conformational changes induced by the effectors on the pyrophosphorylase, resulting in an enzyme with different stability to heat treatments.