Epitopic regions recognized by monoclonal antibodies against rat brain hexokinase: association with catalytic and...

Using direct and competitive epitope mapping methods, 23 monoclonal antibodies (Mabs) against rat brain hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) were divided into nine groups, each recognizing epitopes within defined surface regions of the N- or C-terminal domains; the latter have been associated with regulatory or catalytic functions, respectively. Reactivity of Mabs with the isolated domains was also studied. Based on the effect of various ligands on immunoreactivity, specific regions involved in ligand-induced conformational changes were identified. Adjacent epitopic regions, designated Regions F and G and located in the N- and C-terminal domains, respectively, were selectively affected by inhibitory hexose 6-phosphates (or analogs), marking these regions as being involved in transmission of the conformational signal from the regulatory N-terminal domain to the catalytic C-terminal domain. Consistent with this, the Ki for inhibition of the enzyme by the glucose 6-phosphate analog, 1,5-anhydroglucitol-6-phosphate, was markedly increased by Mabs binding in these regions, but unaffected by Mabs binding elsewhere in the molecule. Reactivity with Mabs recognizing conformationally sensitive epitopes in Region H of the C-terminal domain was greatly decreased by binding of substrate hexoses that induce closure of a cleft in the catalytic domain; selective recognition of the "open cleft" conformation, thereby preventing closure of the cleft required for progression of the catalytic cycle, can account for the marked decrease in Vmax that results from binding of these Mabs. Reactivity with Mabs binding to Region H was also decreased in the presence of inhibitory hexose 6-phosphates, implying that cleft closure was also induced by the latter; this is consistent with the suggestion that limitation of access to the C-terminal ATP binding site, resulting from cleft closure, is a factor in inhibition of the enzyme.