Thr680 is a critical residue in the active site of HK and is highly conserved among hexokinases from other species.

(A) Amino acid alignment of the region surrounding human HK-I residue Thr680 among 30 hexokinases. Where applicable, specific isozymes are denoted. Conserved residues compared with human HK-I are in shaded gray. The “c” in parentheses refers to the C-terminal domain of HK. (B) Schematic drawing of the active site of HK-I shows Thr680, bound glucose, and adenosine diphosphate (ADP) in ball-and-stick representation (atom coloring: carbon, black; oxygen, red; nitrogen, blue; and phosphor, magenta). The carbon atom that is absent in the Thr680Ser mutant is colored gray. Illustrated is the central position occupied by Thr680 in the active site where it is located at the tip of a loop and makes a hydrogen bond with the α-phosphoryl group of ADP. This figure was generated from the atomic coordinates of the HK-I ADP/glucose complex7 (protein data bank entry 1DGK) using computer programs Molscript22 and Raster3D.23