Long chain fatty acids inhibit and medium chain fatty acids activate mammalian cardiac hexokinase

We investigated the effect of non-esterified fatty acids (FAs) on bovine heart hexokinase (type I: ATP: -hexose 6-phosphotransferase, EC 2.7.1.1). Long chain FAs (C14 to C20) inhibited the enzyme in a way that correlated positively with both the chain length and the degree of unsaturation. Medium chain FA with 12 or less carbons activated hexokinase in a chain length dependent manner with the greater activation shown by laurate. The activation constant of laurate was 91.5 μM with a maximal activation of 60.3%. Oleate caused a maximal decrease in specific activity of 25% with an inhibition constant of 79 μM. Using the fluorescent probe cis-parinarate, we found a saturable binding site with Kd of 3.5 μM. Oleate competed the fluorescent probe from the protein with a Kd of 1.4 μM. Medium chain FAs did not compete the probe from HK. The binding of fatty acid to the protein appears to be entropically driven as indicated by an Arrhenius analysis (ΔS=+231.6 J mol−1 deg−1). The presence of oleate significantly increased the KATPm from 0.47 mM to 0.89 mM while the Kglucosem in the presence of the FA (0.026±0.003 mM) was not significantly different from the control (0.014±0.004 mM). A decrease in Vmax values in the presence of oleate indicated that a mixed allosteric inhibition was operating.