High resolution crystal structures of yeast hexokinase complexes with substrates, activators, and inhibitors. Evidence for an allosteric control site.

The structures of both monomeric and dimeric yeast hexokinase complexed with sugar and nucleotide substrates and analogues have been determined at 3.5 to 2.7 b resolution. The most striking finding is that there are two kinds of nucleotide binding sites in the dimeric enzyme. We conclude that one kind of site, the A site, binds the ATP involved in the kinase reaction while the other site, the I site, lies between the two subunits and is an allosteric activator site. AMP and other inhibitors that are competitive with respect to ATP bind at one A site per monomer or two per dimer. A model for an ATP molecule has been built into this site by fitting the adenosine moiety to the AMP difference electron density map at 3.5 A resolution and the y-phosphate into the density of a bound sulfate molecule. This ATP molecule is in a completely extended conformation with the phosphorus of its y-phosphate lying about 6 A from the 6hydroxyl of glucose. An ATP analogue, adenyl-5’-yl p,y-imidodiphosphate, binds in an extended conformation to the I site that lies between the two subunits. There is only one such site per dimer being formed by different side chains from the two subunits. Since the y-phosphate of the ATP molecule in this intersubunit site is more than 20 A from the 6-hydroxyl of either of the 2 glucose molecules, it cannot be directly involved in phosphoryl transfer. These crystal binding studies together with previous studies on the enzyme in solution suggest that the activation of hexokinase by ATP (and other anionic metabolites) observed by Kosow and Rose (Kosow, D. P., and Rose, I. A. (1971)J. Biol. Chem. 246, 2618-2625) could be explained by ATP binding at the I site, resulting in dimerization of the enzyme and stabilization of a more active enzyme conformation. Glucose and sugar inhibitors all bind to the same single site per subunit in both the monomer and the dimer. Both glucose and the inhibitor o-toluoylglucosamine bind with their sugar moieties in the same orientation and configuration. The glucopyranose is in a C-l (chair equatorial) con-