The activity of the ADP-dependent glucokinase from Thermococcus litoralis (TlGK) relies on the highly conserved motifs NXXE (i.e. Asn-Xaa-Xaa-Glu) and HXE (i.e. His-Xaa-Glu). Site-directed mutagenesis of residues Glu279 (HXE) and Glu308 (NXXE) leads to enzymes with highly reduced catalytic rates. The replacement of Glu308 by Gln increased the KM for MgADP- and was activated by free Mg2+. On the other hand, HXE mutants did not affect the KM for MgADP-, were still inhibited by free Mg2+, and caused a large increase on KM for glucose and an 87-fold weaker binding of glucose onto the non-hydrolysable TlGK·AMP-AlF3 complex. Our findings put forward the fundamental role of the HXE motif in glucose binding during ternary complex formation.
Abarca-Lagunas, M. J., Rivas-Pardo, J. A., Ramírez-Sarmiento, C. A., & Guixé, V. (2015). Dissecting the functional roles of the conserved NXXE and HXE motifs of the ADP-dependent glucokinase from Thermococcus litoralis. FEBS Letters, 589(21), 3271–3276. https://doi.org/10.1016/j.febslet.2015.09.013