An ATP binding site model for human brain hexokinase was established based on the similarity of the ATP-binding domains among actin, heat shock protein, glycerol kinase and brain hexokinase. Site directed mutagenesis was performed on the residues in the putative ATP-binding site in order to substantiate or refute the proposed model. The k cat values decreased 1000- and 200-fold for the Asp532Lys and Asp532Glu mutants, 114- and 12-fold for the Arg539Ile and Arg539Lys mutants, 2000- and 2-fold for the Thr680Val and Thr680Ser mutants, respectively. It is suggested that these residues are important for catalysis by either keeping the [gamma]-phosphoryl group of ATP in the correct orientation for catalysis or stabilizing the transition state of the reaction. The mutant Gly534Ala exhibited a 4000-fold decrease in its k catvalue. It appears that the small side chain of Gly534 is important to provide space for ATP binding in the correct orientation. The Gly679Ala mutant did not show any significant change in kinetic parameters, whereas the Gly679Ile mutant was overexpressed as an insoluble protein. Thus, Gly679 is probably responsible for correct folding of the enzyme;Crystals for human brain hexokinase complexed with inorganic phosphate, glucose, or glucose-6-phosphate were obtained. The ternary complex of human brain hexokinase-ATP-glucose was also crystallized. X-ray diffraction data has been collected. The solution of the three dimensional structure is currently being carried out;Minihexokinase was overexpressed in Escherichia coli BL21(DE3) strain and purified to homogeneity. Minihexokinase lost its ability to ameliorate inhibition of glucose-6-P-inhibited mini-hexokinase in the presence of phosphate (Pi). These findings suggest that the Pi site either resides in the N-terminal half of hexokinase or requires the N-terminal portion of the enzyme;The purification procedure for human brain hexokinase was improved, producing 10-fold more protein in half the time.