Role of Mg2+ in the structure and activity of maize (Zea mays L.) isocitrate lyase: indications for hysteretic behaviour.
The role of Mg2+ in the structure and activity of maize isocitrate lyase has been studied by CD, limited proteolysis, protection by ligands against inactivation, and activity measurements at various metal concentrations. From CD and trypsinolysis experiments, the existence of high-affinity binding sites for Mg2+ was demonstrated, and a KdME of 200 microM was determined. Both free enzyme (E) and enzyme molecules with high-affinity sites occupied (ME) are catalytically competent, the former showing 40% of the activity of the latter. Mg2+ thus acts as a non-essential activator. A second Mg2+-binding site with a KdMEM of 6 mM was revealed from protection experiments by increasing Mg2+ concentrations against inactivation. From activity measurements at different Mg2+ concentrations, the affinity of the enzyme for the Mg2+-isocitrate complex (MI) was determined to be KdE(MI) = 9 microM. Maize isocitrate lyase was shown to display hysteretic behaviour. Filling the low-affinity binding sites with Mg2+ induces a conformational change in the high-affinity binding sites resulting in an even higher affinity for Mg2+ (KdME* = 40 microM). On lowering the Mg2+ concentration again, the enzyme only responds slowly: the time needed for all enzyme molecules to return to the conformation at which KdME is 200 microM was found to be 60 min. Finally it was shown that the high-affinity binding site for Mg2+ is not formed at low (4 degrees C) temperature.
Beeckmans
S
S
Department of Protein Chemistry, Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint-Genesius-Rode, Belgium.
Khan
A S
AS
Van Driessche
E
E
D002384:Catalysis; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004795:Enzyme Stability; D007522:Isocitrate Lyase / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism* / Q000494:pharmacology; D011485:Protein Binding; D013696:Temperature; D014357:Trypsin / Q000378:metabolism; D003313:Zea mays / Q000201:enzymology*
D014357:Trypsin; D007522:Isocitrate Lyase; D008274:Magnesium