Catalytic activation of human glucokinase by substrate binding - Residue contacts involved in the binding of D-glucose to the super-open form and conformational transitions

Abstract

α-D-Glucose activates glucokinase (EC 2.7.1.1) on its binding to the active site by inducing a global hysteretic conformational change. Using intrinsic tryptophan fluorescence as a probe on the α-D-glucose induced conformational changes in the pancreatic isoform 1 of human glucokinase, key residues involved in the process were identified by site-directed mutagenesis. Single-site W→F mutations enabled the assignment of the fluorescence enhancement (ΔF/F0) mainly to W99 and W167 in flexible loop structures, but the biphasic time course of ΔF/F0 is variably influenced by all tryptophan residues. The human glucokinase-α-D-glucose association (Kd = 4.8 ± 0.1 mm at25 °C) is driven by a favourable entropy change (ΔS = 150 ± 10 J·mol -1·K-1). Although X-ray crystallographic studies have revealed the α-D-glucose binding residues in the closed state, the contact residues that make essential contributions to its binding to the super-open conformation remain unidentified. In the present study, we combined functional mutagenesis with structural dynamic analyses to identify residue contacts involved in the initial binding of α-D-glucose and conformational transitions. The mutations N204A, D205A or E256A/K in the L-domain resulted in enzyme forms that did not bind α-D-glucose at 200 mM and were essentially catalytically inactive. Our data support a molecular dynamic model in which a concerted binding of α-D-glucose to N204, N231 and E256 in the super-open conformation induces local torsional stresses at N204/D205 propagating towards a closed conformation, involving structural changes in the highly flexible interdomain connecting region II (R192-N204), helix 5 (V181-R191), helix 6 (D205-Y215) and the C-terminal helix 17 (R447-K460). © 2008 The Authors.