Mutational analysis of allosteric activation and inhibition of glucokinase

Bogumil Zelent, Stella Odili, Carol Buettger, Dorothy K. Zelent, Pan Chen, Deborah Fenner, Joseph Bass, Charles Stanley, Monique Laberge, Jane M. Vanderkooi, Ramakanth Sarabu, Joseph Grimsby, Franz M. Matschinsky*

*Corresponding author for this work

Research output: Contribution to journalArticle

21 Scopus citations

Abstract

GK (glucokinase) is activated by glucose binding to its substrate site, is inhibited by GKRP (GK regulatory protein) and stimulated byGKAs(GK activator drugs). To explore further themechanisms of these processes we studied pure recombinant human GK (normal enzyme and a selection of 31 mutants) using steadystate 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.

Original languageEnglish (US)
Pages (from-to)203-215
Number of pages13
JournalBiochemical Journal
Volume440
Issue number2
DOIs
StatePublished - Dec 1 2011

Keywords

  • Enzyme kinetics
  • Fluorescence quantum yield
  • Glucokinase (GK)
  • Glucokinase activator drug (GKA)
  • Glucokinase mutant (GK mutant)
  • Glucokinase regulatory protein (GKRP)
  • Glucose
  • Tryptophan fluorescence (TF)

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint Dive into the research topics of 'Mutational analysis of allosteric activation and inhibition of glucokinase'. Together they form a unique fingerprint.

  • Cite this

    Zelent, B., Odili, S., Buettger, C., Zelent, D. K., Chen, P., Fenner, D., Bass, J., Stanley, C., Laberge, M., Vanderkooi, J. M., Sarabu, R., Grimsby, J., & Matschinsky, F. M. (2011). Mutational analysis of allosteric activation and inhibition of glucokinase. Biochemical Journal, 440(2), 203-215. https://doi.org/10.1042/BJ20110440