RESUMEN
6-Phosphofructo-1-kinase (PFK) and aldolase are two sequential glycolytic enzymes that associate forming heterotetramers containing a dimer of each enzyme. Although free PFK dimers present a negligible activity, once associated to aldolase these dimers are as active as the fully active tetrameric conformation of the enzyme. Here we show that aldolase-associated PFK dimers are not inhibited by clotrimazole, an antifungal azole derivative proposed as an antineoplastic drug due to its inhibitory effects on PFK. In the presence of aldolase, PFK is not modulated by its allosteric activators, ADP and fructose-2,6-bisphosphate, but is still inhibited by citrate and lactate. The association between the two enzymes also results on the twofold stimulation of aldolase maximal velocity and affinity for its substrate. These results suggest that the association between PFK and aldolase confers catalytic advantage for both enzymes and may contribute to the channeling of the glycolytic metabolism.
Asunto(s)
Fructosa-Bifosfato Aldolasa/metabolismo , Glucólisis , Fosfofructoquinasa-1/metabolismo , Regulación Alostérica , Animales , Antifúngicos/metabolismo , Catálisis , Clotrimazol/metabolismo , Dimerización , Fructosa-Bifosfato Aldolasa/antagonistas & inhibidores , Fructosa-Bifosfato Aldolasa/química , Músculo Esquelético/enzimología , Fosfofructoquinasa-1/antagonistas & inhibidores , Fosfofructoquinasa-1/química , Conformación Proteica , Conejos , Espectrometría de FluorescenciaRESUMEN
Clotrimazole (CTZ) has been proposed as a potential anti-neoplastic agent, which inhibits glucose metabolism. The present work aimed to evaluate the effects of CTZ on the kinetic mechanism of 6-phosphofructo-1-kinase (PFK). We show that CTZ promotes a dose-dependent inhibition of PFK, presenting a K(i) of 28 +/- 2 microM. Inhibition occurs through the dissociation of the enzyme tetramers, as demonstrated through fluorescence spectroscopy and gel filtration chromatography. Moreover, the affinities of the enzyme for ATP and fructose-6-phosphate are reduced 50% and 30%, respectively. Furthermore, the affinity of PFK for ATP at the inhibitory site becomes 2-fold higher. Altogether, the results presented here suggest that PFK inhibition by CTZ involves a decrease in the affinity of PFK for its substrates at the catalytic site with the concomitant potentiation of the inhibitory properties of ATP.