Abstract:
:Slow conformational changes have been proposed to be responsible for the kinetic positive cooperativity of glucokinase (GK) with glucose. Induced-fit and preexisting equilibrium kinetic models have been previously suggested. In the present study, equilibrium and pre-steady-state fluorescence spectroscopy has been used to resolve those conflicting reports. Multiphasic transients were observed after rapid mixing of apo-GK with glucose. Progress curve analysis revealed inconsistencies with the induced-fit model. The glucose dependence of the major kinetic phase supported the preexistence of at least two slowly interconverting GK conformers. In the absence of glucose, approximately 80% of the GK population is likely poised in a largely open conformation (K(D) approximately 30 mM). The remaining 20% is in a more compact conformation (K(D) approximately 0.2 mM). Transients revealed three additional phases likely reflecting intermediates on the pathway between the superopen and the closed conformer. Using an intrinsically fluorescent GK activator (GKA), it was shown that GK can bind GKA in the absence of glucose, confirming the validity of the preexisting equilibrium model. Additionally, a perturbation of the GKA binding kinetic parameters after preequilibration of closed GK with an ATP analogue suggested a local rearrangement of the allosteric site upon nucleotide binding. Our data suggest that, in the absence of any ligand, GK might be able to extensively sample the conformational space delimited by the superopen and the closed conformations. The complex GK conformational equilibrium is readily shifted upon binding of ligands such as glucose or GKAs on specific GK conformers.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Antoine M,Boutin JA,Ferry Gdoi
10.1021/bi900374csubject
Has Abstractpub_date
2009-06-16 00:00:00pages
5466-82issue
23eissn
0006-2960issn
1520-4995journal_volume
48pub_type
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