Crystallographic studies and model building of ATP at the active site of hexokinase

Abstract

The structure of a complex of 8-bromoadenosine monophosphate with hexokinase in crystal form BIII has been determined from a 3 Å resolution difference electron density map. The ribose is anti relative to the adenine moiety, and the sugar pucker is C-2′-endo. In a model constructed to fit this map, the adenine is situated in a shallow depression on the surface of the large lobe of the enzyme at the entrance to its deep cleft. Atom N-7 is hydrogen-bonded to the backbone carbonyl oxygen of residue 396. The 2′ and 3′-ribose hydroxyls are hydrogen-bonded to Asx317 and Ser319, respectively. There is no difference electron density corresponding to the phosphate. The three phosphates of ATP and the essential metal ion have been model built into the active site using the known crystal structure of tripolyphosphato tetraamine cobalt (III) (Merritt et al., 1978), the position of 8-bromoadenosine monophosphate, and the position of a sulfate ion bound to these crystals. In our model for ATP the β and γ-phosphates are hydrogen-bonded by Ser393 and a backbone nitrogen of Ser212, and the cobalt ion interacts with Ser212. In the closed conformation of hexokinase which is induced by the binding of glucose, model building suggests there would be additional contacts between the small enzyme lobe and the metal ion. This could explain the observed synergism in ATP and glucose binding to hexokinase in solution. Since the γ-phosphate of the ATP in our model is nearly 6 Å from the 6-hydroxyl of glucose, an additional conformational change must occur in the enzyme upon formation of the ternary complex.