The allosteric regulation of hexokinase C from amphibian liver.
A type C hexokinase (ATP:D-hexose-6-phosphotransferase EC 2.7.1.1) was partially purified from the liver of the frog Calyptocephalella caudiverbera. The enzyme is inhibited by glucose levels in the range of normal blood sugar concentrations. The extent of the inhibition by glucose depends on the concentration of ATP, being most marked between 1 and 5 mM ATP. Fructose, although a substrate, was not inhibitory of its own phosphorylation. The inhibitory effect of high glucose levels exhibited a strong, reversible pH dependence being most marked at pH 6.5. At pH 7.5 the inhibition by high glucose levels was a function of the enzyme concentration, the effect being stronger at high enzyme concentrations, whereas no inhibition was observed when assaying very diluted preparations. At all enzyme concentrations studied, high levels of glucose caused no inhibition at pH 8.5, whereas at pH 6.5 strong inhibition was always observed. Short times of photooxidation of hexokinase C as well as incubation with low concentrations of p-chloromercuribenzoate resulted in the loss of the inhibition by excess of glucose. Glucose-6-phosphate was found to be a strong inhibitor of hexokinase C but only at high glucose levels. The inhibitory effect of glucose-6-P follows sigmoidal kinetics at low (about 0.02 mM) glucose concentrations, the Hill coefficient being 2.3. The kinetics of the inhibition became hyperbolic at high (greater than 0.2 mM) glucose levels. These results suggest that the inhibition of hexokinase C by excess glucose is due to the interaction of glucose with a second, aldose-specific, regulatory site on the enzyme. The modification of the inhibitory effect by ATP, glucose-6-P, enzyme concentration, and pH, all of them at physiological levels, indicates a major role for hexokinase C in the regulation of glucose utilization by the liver.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000494:Allosteric Regulation; D000818:Animals; D002729:Chloromercuribenzoates / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D006593:Hexokinase / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008099:Liver / Q000201:enzymology*
D002729:Chloromercuribenzoates; D007527:Isoenzymes; D000255:Adenosine Triphosphate; D006593:Hexokinase; D005947:Glucose