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

Abstract

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