ATP-BINDING SITE OF HUMAN BRAIN HEXOKINASE AS STUDIED BY MOLECULAR MODELING AND SITE-DIRECTED MUTAGENESIS

The interaction of ATP with the active site of hexokinase is unknown s ince the crystal structure of the hexokinase-ATP complex is unavailabl e. It was found that the ATP binding site of brain hexokinase is homol ogous to that of actin, heat shock protein hsc70, and glycerol kinase. On the basis of these similarities, the ATP molecule was positioned i n the catalytic domain of human brain hexokinase, which was modeled fr om the X-ray structure of yeast hexokinase. Site-directed mutagenesis was performed to test the function of residues presumably involved in interaction with the tripolyphosphoryl moiety of ATP. Asp532, which is thought to be involved in binding the Mg2+ ion of the MgATP(2-) compl ex, was mutated to Lys and Glu. The k(cat) values decreased 1000- and 200-fold, respectively, for the two mutants. Another residue, Thr680 w as proposed to interact with the gamma-phosphoryl group of ATP through hydrogen bonds and was mutated to Val and Ser. The k(cat) value of th e Thr680Val mutant decreased 2000-fold, whereas the k(cat) value of th e Thr680Ser decreased only 2.5-fold, implying the importance of the hy droxyl group. The K-m and dissociation constant values for either ATP or glucose of all the above mutants showed little or no change relativ e to the wild-type enzyme. The K-i values for the glucose 6-phosphate analogue 1,5-anhydroglucitol 6-phosphate, were the same as that of the wild-type enzyme, and the inhibition was reversed by inorganic phosph ate (P-i) for all four mutants. The circular dichroism spectra of the mutants were the same as that of the wild-type enzyme. The results fro m the site-directed mutagenesis demonstrate that the presumed interact ions of investigated residues with ATP are important for the stabiliza tion of the transition state.