380 Biochimica et Biophysica Acta, 705 ( i 982) 380-384 Elsevier Biomedical Press BBA 31249 THE 6-HYDROXYMETHYL GROUP OF A HEXOSE IS ESSENTIAL FOR THE SUBSTILATE- INDUCED CLOSURE OF THE CLEFT IN HEXOKINASE MENAHEM SHOHAM * and THOMAS A. STEITZ ** Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06511 (U.S.A.) (Received January 19th, 1982) Key words: Hydroxymethyl group; Hexose substrate; Hexokinase structure," Cleft closure Yeast hexokinase B (ATP:-hexose 6-phosphotransferase, EC 2.7.1.1) was crystallized in the presence of D-xylose and ADP, and its structure was determined at 7 J, resolution. The enzyme is in the 'open' conformation which is characteristic of the enzyme crystallized in the absence of glucose, rather than in the 'closed' conformation that is observed with the glucose complex. That is, the binding of xylose into the large cleft that separates the molecule into two lobes does not cause the cleft to close. We conclude, then, that the glucose 6-hydroxymethyl group (which binds to an aspartic acid and a serine) is essential for the hexose-induced conformational change. Introduction Yeast hexokinase (ATP:D-hexose 6-phos- photransferase, EC 2.7.1.1) exists in a number of distinct conformational states during the couse of catalysis [1-9]. It can exist as an active monomer or an active dimer which in the course of catalysis forms binary complexes with sugar or ATP.sub- strates and then forms ternary complexes with both sugar and nucleotide substrates (Fig. 1). Phosphoryl transfer occurs only when both sub- strates are bound to the enzyme. Different hexokinase conformers have been trapped in crystalline lattices and their structure determined. The monomeric apoenzyme has been crystallographically refined at 2.1 ,A resolution [ 1- 3]. The dimer has been crystallized in the absence of sugars and its structure determined at 3.5 ,A resolution [4-6]. The structure of hexokinase crys- * Permanent address: Department of Structural Chemistry, The Weizmann Institute of Science, Rehovot, Israel. ** To whom correspondence should be addressed. Abbreviation: Mops, 4-morpholinepropanesulfonicacid. 0167-4838/82/0000-0000/$02075 © 1982 Elsevier Biomedical Press tallized in the presence of glucose shows that glucose binding causes a large conformational change on the enzyme [7]. One lobe is rotated by 12 ° relative to the other, as compared to the apoenzyme, thereby closing off the cleft and 'bury- ing' the glucose substrate [8,9]. The other sub- strate, ATE can be diffused into crystals of the \Ey ° ~GLUC. AlP - E", C~JC. E~------.E', 6-P + A~ Fig. 1. Steps in the reaction pathway of the yeast hexokinase monomer, E, which can form an enzymatically active dimer E 2. The preferred pathway is glucose adding first to form E'. glucose. E', E" and E" represent conformational alterations induced by ligand binding. The crystal structures of E [2], E 2 [6] and E' [9] have been determined at high resolution, and the structure of E" is reported here at low resolution.