Ligand-induced conformations of rat brain hexokinase: Studies with hexoses, hexose 6-phosphates, and nucleoside triphosphates leading to development of a new model for the enzyme

Abstract

Various ligands of rat brain hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) have been found to protect the enzyme against either (or both) chymotryptic digestion or inactivation by glutaraldehyde. Using this protective effect, the K d for various enzyme-ligand complexes has been estimated: hexokinase-Glc, K d = 0.24 ± 0.03mM (chymotryptic digestion), K d = 0.26 ± 0.07mM (glutaraldehyde inactivation); hexokinase-Glc-6- P, K d = 0.041 ± 0.005m M (glutaraldehyde inactivation); hexokinase-ATP, K d = 1.01 ± 0.28mM (chymotryptic digestion); hexokinase-ATP-Mg 2+, K d = 0.07-0.08mM (chymotryptic digestion). Other nucleoside triphosphates (UTP, ITP, GTP, and CTP) were much less effective than ATP at protecting against chymotrypsin. Various hexoses were tested for their ability to protect against glutaraldehyde. Only ‛good” substrates (mannose, 2-deoxyglucose) protected; nonsubstrates (galactose, arabinose) and N-acetylglucosamine, a competitive inhibitor of Glc binding, were not effective. Various hexose 6-phosphates were tested for their ability to protect against glutaraldehyde inactivation. Glc-6- P was much more effective than were mannose-6- P, galactose-6- P, or fructose-6- P. It was observed that ‛good” substrates (Glc, mannose) increased the effectiveness of Glc-6- P at solubilizing the mitochondrial form of the enzyme; galactose and N-acetylglucosamine had no effect on solubilization by Glc-6- P. These results are taken as an indication of enhanced Glc-6- P binding in the presence of Glc, as previously reported by Ellison et al. ( J. Biol. Chem., 250, 1864–1871, 1975) . Along with previous studies on ligand-induced conformations and kinetics of this enzyme, these results form the basis for a new model for brain hexokinase. This model specifically takes into account the ligand-induced conformations at various points in the catalytic cycle and specifically accounts for the ability of various hexoses to serve as substrates and hexose 6-phosphates to serve as inhibitors in terms of their ability to induce specific conformations of the enzyme. The properties of the various conformations involved in the model are designated by a four-letter code which facilitates comparison and discussion.