Glucose Modulation of Glucokinase Activation by Small Molecules

Abstract

Small molecule activators of glucokinase (GK) were used in kinetic and equilibrium binding studies to probe the biochemical basis for their allosteric effects. These small molecules decreased the glucose K 0.5 ( approximately 1 mM vs approximately 8 mM) and the glucose cooperativity (Hill coefficient of 1.2 vs 1.7) and lowered the k cat to various degrees (62-95% of the control activity). These activators relieved GK's inhibition from glucokinase regulatory protein (GKRP) in a glucose-dependent manner and activated GK to the same extent as control reactions in the absence of GKRP. In equilibrium binding studies, the intrinsic glucose affinity to the activator-bound enzyme was determined and demonstrated a 700-fold increase relative to the apoenzyme. This is consistent with a reduction in apparent glucose K D and the steady-state parameter K 0.5 as a result of enzyme equilibrium shifting to the activator-bound form. The binding of small molecules to GK was dependent on glucose, consistent with the structural evidence for an allosteric binding site which is present in the glucose-induced, active enzyme form of GK and absent in the inactive apoenzyme [Kamata et al. (2004) Structure 12, 429-438]. A mechanistic model that brings together the kinetic and structural data is proposed which allows qualitative and quantitative analysis of the glucose-dependent GK regulation by small molecules. The regulation of GK activation by glucose may have an important implication for the discovery and design of GK activators as potential antidiabetic agents.

Biophysical Characterization of the Interaction between Hepatic Glucokinase and Its Regulatory Protein

“…Our ITC and SPR data demonstrate that prototypical GKAs of this class prevent binding of GKRP to GK. This observation is in line with recently published biochemical data on the effect of related GKAs (53). We found this impairment of the GK-GKRP association also for a compound (GKA1) that was previously suggested not to interfere with this protein-protein interaction (15).…”

Research and Development of Glucokinase Activators for Diabetes Therapy: Theoretical and Practical Aspects

“…4) (Dunten et al 2004;Heredia et al 2006;Ralph et al 2008;Zelent et al 2008). In fact some GKAs do not bind at all in the absence of a sugar ligand.…”

“…This GKA interference with GKRP inhibition of glucokinase finds its corollary in the large impact of activating mutations on the apparent Ki of GKRP which may increase maximally more than 100-fold depending on the nature of the mutation. Quantitative comparisons of published results of in vitro studies of different compounds are complicated by the fact that their K d and EC 50 values are inversely related to the glucose levels and may vary as much as 10-100 fold, respectively, in comparing low and high glucose saturation (Ralph et al 2008). In order to overcome this difficulty, the relative GK activity index (%-AI) could be used to compare the efficacy of different GKAs.…”

The genetic basis of congenital hyperinsulinism

“…All reported activating mutations cluster in a region of the enzyme, which has been termed the allosteric activator site and is remote to the substrate binding site. The allosteric site of GCK is where small molecule activators bind, suggesting a critical role of the allosteric site in the regulation of GCK activity 92. Both GCK activators and activating mutations increase enzyme activity by enhancing the affinity for glucose as described by a decrease in K 0.5 93.…”