Figure 3

Phosphoryl transfer mechanism found in the Group 4 kinases (hexokinase family with polyol substrate). This occurs via coordination of an arginine residue to the N7/C8 of the imidazole moiety mediating the change in C8 hybridization from sp2 to sp3 hybridized, and altering the protonation of N7 and C8. Protonation of the N7 occurs via a conserved the Arg residue with the NH₂ being coordinated directly to N7, with an interatomic distance of 3.065 ± 0.823 Å. The Arg residue in Group 4 kinases is always stabilized by an associated Asp/Glu residue. The reaction occurs via a carbene mechanism with the carbene being stabilized via the interaction of a conserved backbone Gly carbonyl that is within bonding distance of C8, causing C8-H to become more acidic, allowing for the protonation of the α-PO₄, via a conserved Ser/Thr. There is a concomitant transfer of an H⁺ from the α-PO₄ to the β-PO₄ via a conserved Arg, thereby facilitating the formation of the pentavalent intermediate between the γ-PO₄ and the substrate nucleophile. There is a concomitant Asp-mediated deprotonation of the substrate -OH, allowing for the nucleophilic attack by the substrate. This creates the pentavalent intermediate and allows phosphoryl transfer. The protonated Asp then transfers the proton to the γ-PO₄, changing the coordination of the Mg²⁺ from being β-PO₄ to γ-PO₄ coordinated to being α-PO₄ to β-PO₄ coordinated. The H⁺ originally arising from the C8 is then transferred back to C8, allowing the electron density of the adenyl moiety to return to the "ground-state" distribution.