Identification of residues mediating homotropic regulation in human hexokinase 3 reveals a common allosteric interface shared with hexokinase 1

Vertebrate hexokinases (HKs) display a variety of allosteric phenomena, including activation and inhibition by both homotropic and heterotropic ligands. The extent to which these homologs share a common allosteric mechanism is unknown. A unique trait of the vertebrate hexokinase 3 (HK3) orthologs is substrate inhibition by glucose. Here, we demonstrate that the isolated, regulatory N-terminal domain of human HK3 contains a low affinity glucose binding site whose dissociation constant (3.2 ± 0.4 mM) approximates the K_i value for glucose (11 ± 2 mM) observed in assays of the full-length enzyme. The isolated, catalytic C-terminal domain harbors a high affinity glucose binding site whose dissociation constant (6.2 ± 0.6 μM) resembles the K_m value for glucose (53 ± 1 μM). Substitution of Asn221 in full-length HK3, which lies within the N-terminal glucose binding site, reduces substrate inhibition by 30-fold while leaving other steady-state kinetic parameters unchanged. Homotropic inhibition of HK3 is largely independent of ATP concentrations, in contrast to heterotropic inhibition of hexokinase 1 (HK1) by glucose 6-phosphate, which is competitive with respect to ATP. Adding a 10-fold molar excess of the N-terminal domain to the C-terminal domain fails to alter substrate inhibition, suggesting that interdomain communication in HK3 requires their physical connection. Disrupting a coulombic interaction between N-terminal residue Asp264 and C-terminal residue Arg807, two conserved residues previously shown to participate in HK1 regulation, attenuates glucose inhibition of HK3. Our data support a common allosteric interface in HK1 and HK3, wherein effector binding at spatially distinct sites within the regulatory N-terminus is communicated to the catalytic C-terminus via conserved coulombic residues.