ROLE OF MG2-MAYS L.) ISOCITRATE LYASE - INDICATIONS FOR HYSTERETIC BEHAVIOR( IN THE STRUCTURE AND ACTIVITY OF MAIZE (ZEA)

The role of Mg2+ in the structure and activity of maize isocitrate lya se has been studied by CD, limited proteolysis, protection by ligands against inactivation, and activity measurements at various metal conce ntrations. From CD and trypsinolysis experiments, the existence of hig h-affinity binding sites for Mg2+ was demonstrated, and a K-dME of 200 mu M was determined. Both free enzyme (E) and enzyme molecules with h igh-affinity sites occupied (ME) are catalytically competent, the form er showing 40% of the activity of the latter. Mg2+ thus acts as a non- essential activator. A second Mg2+-binding site with a K-dMEM, of 6 mM was revealed from protection experiments by increasing Mg2+ concentra tions against inactivation. From activity measurements at different Mg 2+ concentrations, the affinity of the enzyme for the Mg2+-isocitrate complex (MI) was determined to be K-dE(MI) = 9 mu M. Maize isocitrate lyase was shown to display hysteretic behaviour. Filling the low-affin ity binding sites with Mg2+ induces a conformational change in the hig h-affinity binding sites resulting in an even higher affinity for Mg2 (K-dMe = 40 mu M). On lowering the Mg2+ concentration again, the enzy me only responds slowly: the time needed for all enzyme molecules to r eturn to the conformation at which K-dME is 200 mu M was found to be 6 0 min. Finally it was shown that the high-affinity binding site for Mg 2+ is not formed at low (4 degrees C) temperature.