Binding of nucleoside triphosphates, inorganic phosphate, and other polyanionic ligands to the N-terminal region of rat brain hexokinase: relationship to regulation of hexokinase activity by antagonistic interactions between glucose 6-phosphate and inorganic phosphate.
Mg2(+)-chelates of several nucleoside triphosphates were shown to increase the inactivation of rat brain hexokinase (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) by 0.6 M guanidine hydrochloride, with ATP-Mg2+ having the greatest effect; unchelated forms did not significantly affect inactivation. Since catalytic activity has been associated with the C-terminal half of the molecule, these results were interpreted as indicating a destabilization of this C-terminal region by binding of these chelates to the substrate nucleotide sites, with the particular effectiveness of ATP-Mg2+ reflecting the specificity for this species as a phosphoryl donor. These compounds were also shown to bind to the N-terminal half of the enzyme, as judged by their ability to protect against denaturation by guanidine hydrochloride and subsequent digestion with trypsin. Both free and Mg2(+)-chelated forms afforded protection, with the unchelated nucleotides being most effective; a preference for ATP was seen only with the chelated forms. Thus, it was concluded that the N-terminal half of hexokinase contains a relatively nonspecific nucleotide binding site, distinct from the substrate nucleotide site previously shown to reside in the C-terminal half. On the basis of this same ability to protect the N-terminal half against denaturation and proteolysis, several other polyanionic ligands were shown to bind to this region of the molecule. These included inorganic phosphate, its analogs, sulfate and arsenate, and its homologs, pyrophosphate and tripolyphosphate. All of these anionic ligands were also shown to antagonize inhibition by the glucose 6-phosphate (Glc-6-P) analog, 1,5-anhydroglucitol 6-phosphate. The allosteric site for binding of Glc-6-P has previously been shown to reside in the N-terminal half of the molecule, and it is suggested that the antagonism of inhibition by Glc-6-P (or its analog) by these anionic ligands results from interaction with an anion binding site for which the 6-phosphate group of inhibitory hexose 6-phosphates must compete. A model depicting possible relationships between ligand binding sites on brain hexokinase, and how their interactions might lead to observed regulatory properties, is developed based on these and previous studies of ligand binding as well as evidence that mammalian hexokinases (Mr 100,000) have evolved by duplication and fusion of a gene coding for an ancestral hexokinase with Mr 50,000 and which, like the mammalian enzyme, was sensitive to inhibition by Glc-6-P.
White
T K
TK
Department of Biochemistry, Michigan State University, East Lansing 48824.
Wilson
J E
JE
D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D019298:Glucose-6-Phosphate; D005958:Glucosephosphates / Q000378:metabolism*; D006593:Hexokinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006706:Homeostasis; D008961:Models, Structural; D008970:Molecular Weight; D010449:Peptide Mapping; D010710:Phosphates / Q000378:metabolism*; D051381:Rats; D012265:Ribonucleotides / Q000378:metabolism*; D014357:Trypsin
D005958:Glucosephosphates; D010710:Phosphates; D012265:Ribonucleotides; D019298:Glucose-6-Phosphate; D006593:Hexokinase; D014357:Trypsin