Effect of ligands and redox state on phosphofructokinase quaternary structure and enzymatic activity

Abstract Background Alterations in glycolytic flux are characteristic of cancer cells and have recently been linked to Alzheimeru0027s disease. Phosphofructokinase (PFK) activity is a key regulator of glycolysis and also modulates apoptosis. PFK has a complex quaternary structure, with subunits associating into higher order oligomers including tetramers. This study aimed to investigate the influence of potential regulatory factors on the quaternary structure of PFK muscle isoform splice variant 1 (PFK-M1) and the resulting effect on enzyme activity. Methods BL21(DE3) competent Escherichia coli cells were transformed with plasmid pDEST14 containing cDNA for N-terminal His6 tagged human PFK-M1, and induced with isopropyl β-D-1-thiogalactopyranoside. Cells were lysed by liquid homogenisation and purified via immobilised metal ion affinity chromatography. Quaternary structure was explored with size exclusion chromatography. Protein stability was studied with thermal denaturation assays. Activity was determined via enzyme-linked assay (pyruvate kinase–lactate dehydrogenase). Findings PFK-M1 was verified with mass spectrometry (Mascot score 132). PFK activity decreased at a rate inversely proportional to enzyme concentration. Activity after 1 day was 52% of baseline at 320 μg/ml and 9% at 80 μg/mL. Activity after 1 day at 100 μg/mL was partly preserved by reducing agents dithiothreitol (DTT) (27%) and tris (2-carboxyethyl)phosphine (TCEP) (84%), or ligands ATP (75%) and fructose 6-phosphate (F6P) (84%). Combining TCEP and F6P abolished any loss of activity. Size exclusion chromatography showed on-column dissociation and aggregation, with a broad size distribution. Addition of TCEP and F6P confined size distribution to tetrameric state only. Thermal denaturation assays confirmed that stability was proportional to concentration. PFK melting temperature was raised by TCEP (2·9°C) and ATP (4°C); simultaneous use gave a synergistic enhancement (9·3°C). Interpretation PFK-M1 shows time-dependent and concentration-dependent inactivation correlating with changes in enzyme quaternary structure. These changes can be prevented with ligands or reducing agents in a synergistic manner, indicating different mechanisms of action. We propose a model whereby PFK exists in a dynamic equilibrium between different oligomeric states. Substrates stabilise the active tetrameric form, whereas reducing agents prevent formation of inactive aggregations. Funding Wellcome Trust.