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: D-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 microM with a maximal activation of 60.3%. Oleate caused a maximal decrease in specific activity of 25% with an inhibition constant of 79 microM. Using the fluorescent probe cis-parinarate, we found a saturable binding site with K(d) of 3.5 microM. Oleate competed the fluorescent probe from the protein with a K(d) of 1.4 microM. 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 (DeltaS=+231.6 J mol(-1) deg(-1)). The presence of oleate significantly increased the K(ATP)(m) from 0.47 mM to 0.89 mM while the K(glucose)(m) 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 V(max) values in the presence of oleate indicated that a mixed allosteric inhibition was operating.
Stewart
J M
JM
Biochemistry Program, Department of Biology, Mount Allison University, Flemington Building, 63B York St. E4L 1G7, Sackville, NB, Canada. jstewart@mta.ca
Blakely
J A
JA
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D005230:Fatty Acids, Nonesterified / Q000494:pharmacology*; D005231:Fatty Acids, Unsaturated / Q000494:pharmacology; D005456:Fluorescent Dyes; D005947:Glucose / Q000378:metabolism; D006321:Heart / Q000187:drug effects*; D006593:Hexokinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007850:Lauric Acids / Q000494:pharmacology; D009206:Myocardium / Q000201:enzymology; D019301:Oleic Acid / Q000378:metabolism / Q000494:pharmacology; D013696:Temperature
D004791:Enzyme Inhibitors; D005230:Fatty Acids, Nonesterified; D005231:Fatty Acids, Unsaturated; D005456:Fluorescent Dyes; D007850:Lauric Acids; C030358:lauric acid; D019301:Oleic Acid; D000255:Adenosine Triphosphate; D006593:Hexokinase; D005947:Glucose