title: Sugar binding to recombinant wild-type and mutant glucokinase monitored by kinetic measurement and tryptophan fluorescence.
abstract: Tryptophan fluorescence was used to study GK (glucokinase), an enzyme that plays a prominent role in glucose homoeostasis which, when inactivated or activated by mutations, causes diabetes mellitus or hypoglycaemia in humans. GK has three tryptophan residues, and binding of D-glucose increases their fluorescence. To assess the contribution of individual tryptophan residues to this effect, we generated GST-GK [GK conjugated to GST (glutathione transferase)] and also pure GK with one, two or three of the tryptophan residues of GK replaced with other amino acids (i.e. W99C, W99R, W167A, W167F, W257F, W99R/W167F, W99R/W257F, W167F/W257F and W99R/W167F/W257F). Enzyme kinetics, binding constants for glucose and several other sugars and fluorescence quantum yields (varphi) were determined and compared with those of wild-type GK retaining its three tryptophan residues. Replacement of all three tryptophan residues resulted in an enzyme that retained all characteristic features of GK, thereby demonstrating the unique usefulness of tryptophan fluorescence as an indicator of GK conformation. Curves of glucose binding to wild-type and mutant GK or GST-GK were hyperbolic, whereas catalysis of wild-type and most mutants exhibited co-operativity with D-glucose. Binding studies showed the following order of affinities for the enzyme variants: N-acetyl-D-glucosamine>D-glucose>D-mannose>D-mannoheptulose>2-deoxy-D-glucose>>L-glucose. GK activators increased sugar binding of most enzymes, but not of the mutants Y214A/V452A and C252Y. Contributions to the fluorescence increase from Trp(99) and Trp(167) were large compared with that from Trp(257) and are probably based on distinct mechanisms. The average quantum efficiency of tryptophan fluorescence in the basal and glucose-bound state was modified by activating (Y214A/V452A) or inactivating (C213R and C252Y) mutations and was interpreted as a manifestation of distinct conformational states.
authors: Department of Biochemistry and Biophysics and Diabetes Research Center, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors: Bogumil
authors: B
authors: Zelent
authors: Stella
authors: S
authors: Odili
authors: Carol
authors: C
authors: Buettger
authors: Chiyo
authors: C
authors: Shiota
authors: Joseph
authors: J
authors: Grimsby
authors: Rebecca
authors: R
authors: Taub
authors: Mark A
authors: MA
authors: Magnuson
authors: Jane M
authors: JM
authors: Vanderkooi
authors: Franz M
authors: FM
authors: Matschinsky
mesh_terms: D001665:Binding Sites; D002241:Carbohydrates / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D005941:Glucokinase / Q000235:genetics*; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008968:Molecular Conformation; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods; D014364:Tryptophan / Q000737:chemistry*
chemical_list: D002241:Carbohydrates; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014364:Tryptophan; D005982:Glutathione Transferase; D005941:Glucokinase