Cooperative interactions in hexokinase D ("glucokinase"). Kinetic and fluorescence studies.
Hexokinase D, also called hexokinase IV or glucokinase, is the isoenzyme characteristic of liver. In spite of its common name of glucokinase it phosphorylates also other sugars besides glucose; in particular, it phosphorylates fructose with similar specificity to that shown by the other hexokinases. Although hexokinase D is a monomeric protein it displays positive cooperativity with glucose and mannose. In contrast, the kinetic behaviour with 2-deoxyglucose and fructose is Michaelian. Mannose, fructose, 2-deoxyglucose and N-acetylglucosamine are competitive inhibitors of glucose phosphorylation and suppress the cooperativity. The cooperative behaviour can also be suppressed by the presence of glycerol at the assay medium at concentrations over 20%, with a decrease in the K0.5. Neither glycerol nor the inhibitors affect the monomeric state of the enzyme. Hexokinase D exhibits an intrinsic fluorescence at about 326 nm due to tryptophan residues. The binding of glucose to the enzyme enhances the native fluorescence by about 15%. A dissociation constant for glucose of about 3.5 mM was estimated; this value decreased to about 0.5 mM glucose in the presence of glycerol. These and other results are discussed on the basis of steady-state models which assume that hexokinase D exists mainly in one conformation state (EI) in the absence of ligands, and that the binding of glucose or mannose induces a conformational transition to a new conformation EII with higher affinity for the sugar substrates. It is postulated that differences in the velocities of the conformational transitions induced by the different sugar substrates give account of the differences in kinetic behaviour with the different sugar substrates.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D005632:Fructose / Q000378:metabolism; D005941:Glucokinase / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D005990:Glycerol / Q000494:pharmacology; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008358:Mannose / Q000378:metabolism; D008956:Models, Chemical; D013050:Spectrometry, Fluorescence
D005632:Fructose; D000255:Adenosine Triphosphate; D005941:Glucokinase; D005947:Glucose; D005990:Glycerol; D008358:Mannose