Molecular Aspects of Pyrophosphate Regulation of the Activity of Isozymes of Maize Fructose-1,6-Bisphosphate Aldolase
摘要
Two isoenzymes of cytoplasmic aldolase from maize leaves were purified using ion-exchange chromatography. Using a linear NaCl gradient during elution from DEAE-Sephacel column allowed the separation of isoenzymes based on their surface charge. At the concentrations of 101.5 and 122.5 mM sodium chloride, the first and second forms of aldolase were desorbed from the column, respectively. Analysis of kinetic parameters, KM and Vmax, revealed differences in the affinity of aldolase isoenzymes for the substrate. In addition, these enzyme forms were regulated differently at different concentrations of pyrophosphate (PPi). Molecular docking allowed to analyze the type of bonds formed during the interaction of aldolase isoenzymes with pyrophosphate. The ALDO2 protein was characterized by the interaction of Glu144 with pyrophosphate via an additional hydrogen bond. Consequently, the effect on its catalytic function became more pronounced since it can participate in proton transfer during substrate cleavage. The NMR results showed a shift of 1.09 ppm, which may indicate a more significant change in the chemical environment due to the formation of PPi bonds with the amide or carboxyl groups of the amino acids of the active site of ALDO1. NMR and molecular docking results confirm that the aldolase isoenzymes ALDO1 and ALDO2 react differently with pyrophosphate, which is reflected in the regulation of their activity.