Dissecting the functional roles of the conserved NXXE and HXE motifs of the ADP-dependent glucokinase from Thermococcus litoralis

•Mutations of the HXE and NXXE motifs in TlGK lead to decreased catalytic activity.•The NXXE mutant E308Q is activated by free Mg2+ and increases the KM for MgADP−.•HXE mutants are inhibited by free Mg2+ and increase the KM for glucose.•Contrary to previous hypothesis, HXE is involved in glucose binding during ternary complex formation. 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.