Clotrimazole potentiates the inhibitory effects of ATP on the key glycolytic enzyme 6-phosphofructo-1-kinase

Abstract

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 Ki of 28 ± 2 lM. 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.