title: Mutational analysis of allosteric activation and inhibition of glucokinase.
abstract: GK (glucokinase) is activated by glucose binding to its substrate site, is inhibited by GKRP (GK regulatory protein) and stimulated by GKAs (GK activator drugs). To explore further the mechanisms of these processes we studied pure recombinant human GK (normal enzyme and a selection of 31 mutants) using steady-state kinetics of the enzyme and TF (tryptophan fluorescence). TF studies of the normal binary GK-glucose complex corroborate recent crystallography studies showing that it exists in a closed conformation greatly different from the open conformation of the ligand-free structure, but indistinguishable from the ternary GK-glucose-GKA complex. GKAs did activate and GKRP did inhibit normal GK, whereas its TF was doubled by glucose saturation. However, the enzyme kinetics, GKRP inhibition, TF enhancement by glucose and responsiveness to GKA of the selected mutants varied greatly. Two predominant response patterns were identified accounting for nearly all mutants: (i) GK mutants with a normal or close to normal response to GKA, normally low basal TF (indicating an open conformation), some variability of kinetic parameters (k(cat), glucose S(0.5), h and ATP K(m)), but usually strong GKRP inhibition (13/31); and (ii) GK mutants that are refractory to GKAs, exhibit relatively high basal TF (indicating structural compaction and partial closure), usually show strongly enhanced catalytic activity primarily due to lowering of the glucose S(0.5), but with reduced or no GKRP inhibition in most cases (14/31). These results and those of previous studies are best explained by envisioning a common allosteric regulator region with spatially non-overlapping GKRP- and GKA-binding sites.
authors.affiliation: Department of Biochemistry and Biophysics and Diabetes Research Center, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Bogumil
authors.initials: B
authors.lastname: Zelent
authors.forename: Stella
authors.initials: S
authors.lastname: Odili
authors.forename: Carol
authors.initials: C
authors.lastname: Buettger
authors.forename: Dorothy K
authors.initials: DK
authors.lastname: Zelent
authors.forename: Pan
authors.initials: P
authors.lastname: Chen
authors.forename: Deborah
authors.initials: D
authors.lastname: Fenner
authors.forename: Joseph
authors.initials: J
authors.lastname: Bass
authors.forename: Charles
authors.initials: C
authors.lastname: Stanley
authors.forename: Monique
authors.initials: M
authors.lastname: Laberge
authors.forename: Jane M
authors.initials: JM
authors.lastname: Vanderkooi
authors.forename: Ramakanth
authors.initials: R
authors.lastname: Sarabu
authors.forename: Joseph
authors.initials: J
authors.lastname: Grimsby
authors.forename: Franz M
authors.initials: FM
authors.lastname: Matschinsky
mesh_terms: D000494:Allosteric Regulation; D002352:Carrier Proteins; D005453:Fluorescence; D005941:Glucokinase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D017354:Point Mutation; D011487:Protein Conformation; D014364:Tryptophan / Q000737:chemistry
chemical_list: D002352:Carrier Proteins; C080906:glucokinase regulatory protein; D014364:Tryptophan; D005941:Glucokinase; D005947:Glucose