Characterization and Inhibition of Human Hexokinase Domain Containing Protein 1 Reveals an Enzyme with Unique Catalytic and Regulatory Traits

Hexokinase domain containing protein 1 (HKDC1) is a recently discovered fifth human hexokinase isozyme that is significantly upregulated in several disease states, including lung and liver cancers. Cellular studies suggest that HKDC1 is a low activity hexokinase; however, its functional characteristics have remained enigmatic. Here, we describe the kinetic and regulatory features of recombinant human HKDC1, demonstrating it to be a robust hexokinase (<i>k</i>_cat/<i>K</i>_m,glucose = 1.5 × 10⁴ M^-1 s^-1) with a unique glucose <i>K</i>_m value (0.49 ± 0.07 mM) that differs markedly from all other human hexokinase isozymes. The isolated C-terminal domain of HKDC1 displays kinetic characteristics nearly identical to the full-length enzyme, whereas the N-terminal domain is inactive. Unlike all other 100 kDa vertebrate hexokinases characterized to date, HKDC1 is insensitive to product inhibition by physiological concentrations of glucose 6-phosphate, with apparent inhibition constants above 1 mM. The hexokinase activity of HKDC1 is also insensitive to Dinaciclib, a pan cyclin-dependent kinase inhibitor that reportedly disrupts the ability of nuclear localized HKDC1 to phosphorylate retinoblastoma-binding protein 5. Conversely, the hexokinase activity of HKDC1 is potently inhibited by a synthetic glucosamine derivative previously developed for hexokinase 1 and 2, with an IC₅₀ value of 103 ± 6 nM. An HKDC1 variant associated with retinitis pigmentosa, T58M, displays a modest, but statistically significant 2-fold decrease in catalytic efficiency (<i>k</i>_cat/<i>K</i>_m,glucose) compared to the wild-type enzyme. Together, our results provide a detailed functional characterization of recombinant HKDC1 and set the stage for investigating the link between HKDC1 catalysis and human disease.