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.
alpha-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 alpha-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 (DeltaF/F(0)) mainly to W99 and W167 in flexible loop structures, but the biphasic time course of DeltaF/F(0) is variably influenced by all tryptophan residues. The human glucokinase-alpha-D-glucose association (K(d) = 4.8 +/- 0.1 mm at 25 degrees C) is driven by a favourable entropy change (DeltaS = 150 +/- 10 J.mol(-1).K(-1)). Although X-ray crystallographic studies have revealed the alpha-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 alpha-d-glucose and conformational transitions. The mutations N204A, D205A or E256A/K in the L-domain resulted in enzyme forms that did not bind alpha-D-glucose at 200 mm and were essentially catalytically inactive. Our data support a molecular dynamic model in which a concerted binding of alpha-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).
Molnes
Janne
J
Department of Clinical Medicine, University of Bergen, Norway.
Bjørkhaug
Lise
L
Søvik
Oddmund
O
Njølstad
Pål R
PR
Flatmark
Torgeir
T
D001665:Binding Sites; D004789:Enzyme Activation; D005941:Glucokinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D014364:Tryptophan; D005941:Glucokinase; D005947:Glucose