Biochemical basis of glucokinase activation and the regulation by glucokinase regulatory protein in naturally occurring mutations

Glucokinase (GK) has several known polymorphic activating mutations that increase the enzyme activity by enhancing glucose binding affinity and/or by alleviating the inhibition of glucokinase regulatory protein (GKRP), a key regulator of GK activity in the liver. Kinetic studies were undertaken to better understand the effect of these mutations on the enzyme mechanism of GK activation and GKRP regulation and to relate the enzyme properties to the associated clinical phenotype of hypoglycemia. Similar to wild type GK, the transient kinetics of glucose binding for activating mutations follows a general two-step mechanism, the formation of an enzyme-glucose complex followed by an enzyme conformational change. However, the kinetics for each step differed from wild type GK and could be grouped into specific types of kinetic changes. Mutations T65I, Y214C, and A456V accelerate glucose binding to the apoenzyme form, whereas W99R, Y214C, and V455M facilitate enzyme isomerization to the active form. Mutations that significantly enhance the glucose binding to the apoenzyme also disrupt the protein-protein interaction with GKRP to a large extent, suggesting these mutations may adopt a more compact conformation in the apoenzyme favorable for glucose binding. Y214C is the most active mutation (11-fold increase in k(cat)/K(0.5)(h)) and exhibits the most severe clinical effects of hypoglycemia. In contrast, moderate activating mutation A456V nearly abolishes the GKRP inhibition (76-fold increase in K(i)) but causes only mild hypoglycemia. This suggests that the alteration in GK enzyme activity may have a more profound biological impact than the alleviation of GKRP inhibition.     

Glucose modulation of glucokinase activation by small molecules

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.     

Possible Allosteric Significance of Water Structures in Proteins

Abstract

In recent theoretical molecular dynamics studies of ion solvation and transport through the model peptide ionophore, gramicidin A, it has been observed that the waters forming a linear single file within the channel have solvation and dynamic properties quite different from those found in bulk water. Strongly correlated motions among the interior single file column of waters persist over 20 Å. A speculation is entertained that related water structures could provide a mechanism for long range enzymatic allosteric effects as an alternative to chemical action at a distance propagated through the protein itself. Two possible specific mechanisms are discussed, hydraulic and “proton wire”. As a further control mechanism, the possibility is considered of modulating the allosteric effect though protein motion to open or close the channel thus producing a “valve” in the hydraulic line or a “switch” in the proton wire.     

Glucose-induced conformational changes in glucokinase mediate allosteric regulation: transient kinetic analysis

The transient kinetics of glucose binding to glucokinase (GK) was studied using stopped-flow fluorescence spectrophotometry to investigate the underlying mechanism of positive cooperativity of monomeric GK with glucose. Glucose binding to GK was shown to display biphasic kinetics that fit best to a reversible two-step mechanism. GK initially binds glucose to form a transient intermediate, namely, E* x glucose, followed by a conformational change to a catalytically competent E x glucose complex. The microscopic rate constants for each step were determined as follows: on rate k1 of 557 M(-1) s(-1) and off rate k(-1) of 8.1 s(-1) for E* x glucose formation, and forward rate k2 of 0.45 s(-1) and reverse rate k(-2) of 0.28 s(-1) for the conformational change from E* x glucose to E x glucose. These results suggest that the enzyme conformational change induced by glucose binding is a reversible, slow event that occurs outside the catalytic cycle (kcat = 38 s(-1)). This slow transition between the two enzyme conformations modulated by glucose likely forms the kinetic foundation for the allosteric regulation. Furthermore, the kinetics of the enzyme conformational change was altered in favor of E x glucose formation in D2O, accompanied by a decrease in cooperativity with glucose (Hill slope of 1.3 in D2O vs 1.7 in H2O). The deuterium solvent isotope effects confirm the role of the conformational change in the magnitude of glucose cooperativity. Similar studies were conducted with GK activating mutation Y214C at the allosteric activator site that is likely involved in the protein domain rearrangement associated with glucose binding. The mutation enhanced equilibrium glucose binding by a combination of effects on both the formation of E* x glucose and an enzyme conformational change to E x glucose. Kinetic simulation by KINSIM supports the conclusion that the kinetic cooperativity of GK arises from slow glucose-induced conformational changes in GK.     

Optimizing the affinity and specificity of ligand binding with the inclusion of solvation effect

Solvation effect is an important factor for protein–ligand binding in aqueous water. Previous scoring function of protein–ligand interactions rarely incorporates the solvation model into the quantification of protein–ligand interactions, mainly due to the immense computational cost, especially in the structure‐based virtual screening, and nontransferable application of independently optimized atomic solvation parameters. In order to overcome these barriers, we effectively combine knowledge‐based atom–pair potentials and the atomic solvation energy of charge‐independent implicit solvent model in the optimization of binding affinity and specificity. The resulting scoring functions with optimized atomic solvation parameters is named as specificity and affinity with solvation effect (SPA‐SE). The performance of SPA‐SE is evaluated and compared to 20 other scoring functions, as well as SPA. The comparative results show that SPA‐SE outperforms all other scoring functions in binding affinity prediction and “native” pose identification. Our optimization validates that solvation effect is an important regulator to the stability and specificity of protein–ligand binding. The development strategy of SPA‐SE sets an example for other scoring function to account for the solvation effect in biomolecular recognitions. Proteins 2015; 83:1632–1642. © 2015 Wiley Periodicals, Inc.     

A functional link between glucokinase binding to insulin granules and conformational alterations in response to glucose and insulin

Glucokinase (GK) activity is essential for the physiological regulation of insulin secretion by glucose. Because the enzyme exerts nearly total control over glucose metabolism in the beta-cell, even small changes in GK activity exert effects on glucose-stimulated insulin secretion and, consequently, the blood glucose concentration. Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the association of GK with insulin granules is regulated by glucose in the beta-cell. Glucose stimulation increased the rate of fluorescence recovery after photobleaching of GK to insulin granules, indicating that GK is released into the cytoplasm after glucose stimulation. Changes in fluorescence resonance energy transfer between two different fluorescent protein variants inserted on opposing ends of GK were observed after glucose stimulation and correlated with increased enzyme activity. Furthermore, glucose-stimulated changes in GK regulation were blocked by two inhibitors of insulin secretion. Insulin treatment restored GK regulation in inhibited cells and stimulated GK translocation and activation by itself. Together, these data support a model for post-translational regulation of GK whereby insulin regulates both the association of GK with secretory granules and the activity of the enzyme within the pancreatic beta-cell.     

Hydration energy landscape of the active site cavity in cytochrome P450cam

Hydration of protein cavities influences protein stability, dynamics, and function. Protein active sites usually contain water molecules that, upon ligand binding, are either displaced into bulk solvent or retained to mediate protein–ligand interactions. The contribution of water molecules to ligand binding must be accounted for to compute accurate values of binding affinities. This requires estimation of the extent of hydration of the binding site. However, it is often difficult to identify the water molecules involved in the binding process when ligands bind on the surface of a protein. Cytochrome P450cam is, therefore, an ideal model system because its substrate binds in a buried active site, displacing partially disordered solvent, and the protein is well characterized experimentally. We calculated the free energy differences for having five to eight water molecules in the active site cavity of the unliganded enzyme from molecular dynamics simulations by thermodynamic integration employing a three-stage perturbation scheme. The computed free energy differences between the hydration states are small (within 12 kJ mol⁻¹) but distinct. Consistent with the crystallographic determination and studies employing hydrostatic pressure, we calculated that, although ten water molecules could in principle occupy the volume of the active site, occupation by five to six water molecules is thermodynamically most favorable. Proteins 32:381–396, 1998. © 1998 Wiley-Liss, Inc.     

Molecular dynamics simulation,binding free energy calculation and unbinding pathway analysis on selectivity difference between FKBP51 and FKBP52: Insight into the molecular mechanism of isoform selectivity

As co‐chaperones of the 90‐kDa heat shock protein(HSP90), FK506 binding protein 51 (FKBP51) and FK506 binding protein 52 (FKBP52) modulate the maturation of steroid hormone receptor through their specific FK1 domains (FKBP12‐like domain 1). The inhibitors targeting FK1 domains are potential therapies for endocrine‐related physiological disorders. However, the structural conservation of the FK1 domains between FKBP51 and FKBP52 make it difficult to obtain satisfactory selectivity in FK506‐based drug design. Fortunately, a series of iFit ligands synthesized by Hausch et al exhibited excellent selectivity for FKBP51, providing new opportunity for design selective inhibitors. We performed molecular dynamics simulation, binding free energy calculation and unbinding pathway analysis to reveal selective mechanism for the inhibitor iFit4 binding with FKBP51 and FKBP52. The conformational stability evaluation of the “Phe67‐in” and “Phe67‐out” states implies that FKBP51 and FKBP52 have different preferences for “Phe67‐in” and “Phe67‐out” states, which we suggest as the determinant factor for the selectivity for FKBP51. The binding free energy calculations demonstrate that nonpolar interaction is favorable for the inhibitors binding, while the polar interaction and entropy contribution are adverse for the inhibitors binding. According to the results from binding free energy decomposition, the electrostatic difference of residue 85 causes the most significant thermodynamics effects on the binding of iFit4 to FKBP51 and FKBP52. Furthermore, the importance of substructure units on iFit4 were further evaluated by unbinding pathway analysis and residue‐residue contact analysis between iFit4 and the proteins. The results will provide new clues for the design of selective inhibitors for FKBP51.     

Biophysical characterization of the interaction between hepatic glucokinase and its regulatory protein: impact of physiological and pharmacological effectors

Glucokinase (GK) is a key enzyme of glucose metabolism in liver and pancreatic beta-cells, and small molecule activators of GK (GKAs) are under evaluation for the treatment of type 2 diabetes. In liver, GK activity is controlled by the GK regulatory protein (GKRP), which forms an inhibitory complex with the enzyme. Here, we performed isothermal titration calorimetry and surface plasmon resonance experiments to characterize GK-GKRP binding and to study the influence that physiological and pharmacological effectors of GK have on the protein-protein interaction. In the presence of fructose-6-phosphate, GK-GKRP complex formation displayed a strong entropic driving force opposed by a large positive enthalpy; a negative change in heat capacity was observed (Kd = 45 nm, DeltaH = 15.6 kcal/mol, TDeltaS = 25.7 kcal/mol, DeltaCp = -354 cal mol(-1) K(-1)). With k(off) = 1.3 x 10(-2) s(-1), the complex dissociated quickly. The thermodynamic profile suggested a largely hydrophobic interaction. In addition, effects of pH and buffer demonstrated the coupled uptake of one proton and indicated an ionic contribution to binding. Glucose decreased the binding affinity between GK and GKRP. This decrease was potentiated by an ATP analogue. Prototypical GKAs of the amino-heteroaryl-amide type bound to GK in a glucose-dependent manner and impaired the association of GK with GKRP. This mechanism might contribute to the antidiabetic effects of GKAs.     

Role of tyrosine hot‐spot residues at the interface of colicin E9 and immunity protein 9: A comparative free energy simulation study

The endonuclease activity of the bacterial colicin 9 enzyme is controlled by the specific and high‐affinity binding of immunity protein 9 (Im9). Molecular dynamics simulation studies in explicit solvent were used to investigate the free energy change associated with the mutation of two hot‐spot interface residues [tyrosine (Tyr): Tyr54 and Tyr55] of Im9 to Ala. In addition, the effect of several other mutations (Leu33Ala, Leu52Ala, Val34Ala, Val37Ala, Ser48Ala, and Ile53Ala) with smaller influence on binding affinity was also studied. Good qualitative agreement of calculated free energy changes and experimental data on binding affinity of the mutations was observed. The simulation studies can help to elucidate the molecular details on how the mutations influence protein–protein binding affinity. The role of solvent and conformational flexibility of the partner proteins was studied by comparing the results in the presence or absence of solvent and with or without positional restraints. Restriction of the conformational mobility of protein partners resulted in significant changes of the calculated free energies but of similar magnitude for isolated Im9 and for the complex and therefore in only modest changes of binding free energy differences. Although the overall binding free energy change was similar for the two Tyr–Ala mutations, the physical origin appeared to be different with solvation changes contributing significantly to the Tyr55Ala mutation and to a loss of direct protein–protein interactions dominating the free energy change due to the Tyr54Ala mutation. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Probing the dynamics of restriction endonuclease NgoMIV‐DNA interaction by single‐molecule FRET

Many type II restriction endonucleases require two copies of their recognition sequence for optimal activity. Concomitant binding of two DNA sites by such an enzyme produces a DNA loop. Here we exploit single‐molecule Förster resonance energy transfer (smFRET) of surface‐immobilized DNA fragments to study the dynamics of DNA looping induced by tetrameric endonuclease NgoMIV. We have employed a DNA fragment with two NgoMIV recognition sites and a FRET dye pair such that upon protein‐induced DNA looping the dyes are brought to close proximity resulting in a FRET signal. The dynamics of DNA ‐ NgoMIV interactions proved to be heterogeneous, with individual smFRET trajectories exhibiting broadly different average looped state durations. Distinct types of the dynamics were attributed to different types of DNA ‐ protein complexes, mediated either by one NgoMIV tetramer simultaneously bound to two specific sites (“slow” trajectories) or by semi‐specific interactions of two DNA‐bound NgoMIV tetramers (“fast” trajectories), as well as to conformational heterogeneity of individual NgoMIV molecules.     

Structural changes and fluctuations of proteins: I. A statistical thermodynamic model

A general theory of the structural changes and fluctuations of proteins has been proposed based on statistical thermodynanic considerations at the chain level.The “structure” of protein was assumed to be characterized by the state of secondary bonds between unique pairs of specific sites on peptide chains. Every secondary bond changes between the bonded and unboned states by thermal agitation and the “structure” is continuously fluctuating. The free energy of the “structural state” that is defined by the fraction of secondary bonds in the bonded state has been expressed by the bond energy, the cooperative interaction between bonds, the mixing entropy of bonds, and the entropy of polypeptide chains. The most probable “structural state” can be simply determined by graphical analysis and the effect of temperature or solvent composition on it is discussed. The temperature dependence of the free energy, the probability distribution of structural states and the specific heat have been calculated for two examples of structural change.The theory predicts two different types of structural changes from the ordered to disordered state, a “structural transition” and a “gradual structural change” with rising temperature, In the “structural transition”, the probability distribution has two maxima in the temperature range of transition. In the “gradual structural change”, the probability distribution has only one maximum during the change.A considerable fraction of secondary bonds is in the unbonded state and is always fluctuating even in the ordered state at room temperature. Such structural fluctuations in a single protein molecule have been discussed quantitatively.The theory is extended to include small molecules which bind to the protein molecule and affect the structural state. The changes of structural state caused by specific and non-specific binding and allosteric effects are explained in a unified manner.     

Molecular insight into conformational transformation of human glucokinase: conventional and targeted molecular dynamics

Abstract

Glucokinase (GK) plays a key role in the regulation of hepatic glucose metabolism. An unusual mechanism of positive cooperativity of monomeric GK containing only a single binding site for glucose is very interesting and still unclear. The activation process of GK is associated with a large-scale conformational change from the inactive to the active state. Here, conventional and targeted molecular dynamics simulations were used to study the conformational dynamics of GK in the stable configurations and in the transition from active to inactive state. Three phases of the structural reorganization of GK were detected. The first step is a transformation of GK from the active state to the intermediate structure, where the cleft between the domains is open, but alpha helix 13 is still inside the small domain. From this point, there are two alternative paths. One path leads to the inactive state through the release of helix 13 from the inside of small domain to the outside. Other path goes back to the active state. Simulation results reveal the critical role of helix 13 in the transformation of GK from the open state to inactive one and the influence of the loop 2 on the protein transformation between the open and the closed active states. Principal component analysis and covariance matrix analysis were carried out to analyze the dynamics of protein. Importance of hydrogen bonds in the stability of the closed conformation is shown. Overall, our simulations provide new information about the dynamics of GK and its structural transformation.

Communicated by Ramaswamy H. Sarma     

Crystal structure of E339K mutated human glucokinase reveals changes in the ATP binding site

Human glucokinase (GK) plays an important role in glucose homeostasis. An E339K mutation in GK was recently found to be associated with hyperglycemia. It showed lower enzyme activity and impaired protein stability compared to the wild-type enzyme. Here, we present the crystal structure of E339K GK in complex with glucose. This mutation results in a conformational change of His416, spatially interfering with adenosine-triphosphate (ATP) binding. Furthermore, Ser411 at the ATP binding site is phosphorylated and then hydrogen bonded with Thr82, physically blocking the ATP binding. These findings provide structural basis for the reduced activity of this mutant.     

Identification of substrate binding sites in enzymes by computational solvent mapping

Enzyme structures determined in organic solvents show that most organic molecules cluster in the active site, delineating the binding pocket. We have developed algorithms to perform solvent mapping computationally, rather than experimentally, by placing molecular probes (small molecules or functional groups) on a protein surface, and finding the regions with the most favorable binding free energy. The method then finds the consensus site that binds the highest number of different probes. The probe-protein interactions at this site are compared to the intermolecular interactions seen in the known complexes of the enzyme with various ligands (substrate analogs, products, and inhibitors). We have mapped thermolysin, for which experimental mapping results are also available, and six further enzymes that have no experimental mapping data, but whose binding sites are well characterized. With the exception of haloalkane dehalogenase, which binds very small substrates in a narrow channel, the consensus site found by the mapping is always a major subsite of the substrate-binding site. Furthermore, the probes at this location form hydrogen bonds and non-bonded interactions with the same residues that interact with the specific ligands of the enzyme. Thus, once the structure of an enzyme is known, computational solvent mapping can provide detailed and reliable information on its substrate-binding site. Calculations on ligand-bound and apo structures of enzymes show that the mapping results are not very sensitive to moderate variations in the protein coordinates.     

Slow solvation dynamics at the active site of an enzyme: implications for catalysis

Solvation dynamics at the active site of an enzyme, glutaminyl-tRNA synthetase (GlnRS), was studied using a fluorescence probe, acrylodan, site-specifically attached at cysteine residue C229, near the active site. The picosecond time-dependent fluorescence Stokes shift indicates slow solvation dynamics at the active site of the enzyme, in the absence of any substrate. The solvation dynamics becomes still slower when the substrate (glutamine or tRNA(Gln)) binds to the enzyme. A mutant Y211H-GlnRS was constructed in which the glutamine binding site is disrupted. The mutant Y211H-GlnRS labeled at C229 with acrylodan exhibited significantly different solvent relaxation, thus demonstrating that the slow dynamics is indeed associated with the active site. Implications for catalysis and specificity have been discussed.     

Prediction of the binding energy for small molecules,peptides and proteins

A fast and reliable evaluation of the binding energy from a single conformation of a molecular complex is an important practical task. Knowledge‐based scoring schemes may not be sufficiently general and transferable, while molecular dynamics or Monte Carlo calculations with explicit solvent are too computationally expensive for many applications. Recently, several empirical schemes using finite difference Poisson–Boltzmann electrostatics to predict energies for particular types of complexes were proposed. Here, an improved empirical binding energy function has been derived and validated on three different types of complexes: protein–small ligand, protein–peptide and protein–protein. The function uses the boundary element algorithm to evaluate the electrostatic solvation energy. We show that a single set of parameters can predict the relative binding energies of the heterogeneous validation set of complexes with 2.5 kcal/mol accuracy. We also demonstrate that global optimization of the ligand and of the flexible side‐chains of the receptor improves the accuracy of the evaluation. Copyright © 1999 John Wiley & Sons, Ltd.     

Glucokinase in chicken (Gallus gallus). Partial cDNA cloning, immunodetection and activity determination

Chickens are more hyperglycaemic and insulin-resistant than mammals, and in efforts to understand their glucose metabolism we investigated whether glucokinase (GK) is present in chicken liver or pancreas. This enzyme plays a major role in glucose-sensing in mammals and we have examined whether it also contributes to glucose homeostasis in chickens. Using RT-PCR, we cloned and sequenced a partial cDNA fragment (750 bp) from liver and pancreas that showed a high degree of identity with mammalian GK. Using antibodies directed towards human GK, we immunodetected a 50 kDa band in chicken liver and pancreas. The molecular mass of the band and its specific interaction with the antibody suggest that this protein corresponds to a chicken homologue of human GK. We also determined by spectrophotometry a glucokinase-like activity in crude liver homogenates with an apparent half saturating concentration for glucose of 8.6 mM. GK gene and protein expression did not differ between fed and 24 h fasted states but GK-like activity was significantly increased in fed chickens. In conclusion, our study provides evidence for the presence of GK gene and protein in chicken liver and pancreas and shows that the liver enzyme is active.     

Conformational analysis of endothelin-1: Effects of solvation free energy

In order to investigate conformational preferences of the 21-residue peptide hormone endothelin-1 (ET-1), an extensive conformational search was carried out in vacuo using a combination of high temperature molecular dynamics / annealing and a Monte Carlo / minimization search in torsion angle space. Fully minimized conformations from the search were grouped into families using a clustering technique based on rms fitting over the Cartesian coordinates of the atoms of the peptide backbone of the ring region. A wide range of local energy minima were identified even though two disulfide bridges (Cys¹-Cys¹⁵ and Cys³-Cys¹¹) constrain the structure of the peptide. Low energy conformers of ET-1 as a nonionized species in vacuo arestabilized by intramolecular interaction of the ring region (residues 1-15) with the tail (residues 16–21). Strained conformations for individual residues are observed. Conformational similarity to protein loops is established by matching to protein crystal structures In order to assess the influence of aqueous environment on conformational preference, the electrostatic contribution to the solvation energy was calculated for ET-1 as a fully ionized species (Asp⁸, Lys⁹, Glu¹⁰, Asp¹⁸, N- and C-terminus) using a continuum electrostatics model (DelPhi) for each of the conformed generated in vacuo, and the total solvation free energy was estimated by adding a hydrophobic contribution proportional to solvent accessible surface area. Solvation dramatically alters the relative energetics of ET-1 conformers from that calculated in vacuo. Conformers of ET-1 favored by the electrostatic salvation energy in water include conformers with helical secondary structure in the region of residues 9–15. Perhaps of most importance, it was demonstrated that the contribution tosolvation by an individual charge depends not only on its solvent accessibility but on the proximity of other charges, i.e., it is a cooperative effect. This was shown by the calculation of electrostatic solvation energy as afunction of conformation with individual charges systematically turned “on” and “off”. The cooperative effect of multiple charges on solvation demonstrated in this manner calls into question models that relate solvation energysimply to solvent accessibility by atom or residue alone. © 1995 John Wiley & Sons, Inc.     

Insights into mechanism of glucokinase activation: observation of multiple distinct protein conformations

Human glucokinase (GK) is a principal regulating sensor of plasma glucose levels. Mutations that inactivate GK are linked to diabetes, and mutations that activate it are associated with hypoglycemia. Unique kinetic properties equip GK for its regulatory role: although it has weak basal affinity for glucose, positive cooperativity in its binding of glucose causes a rapid increase in catalytic activity when plasma glucose concentrations rise above euglycemic levels. In clinical trials, small molecule GK activators (GKAs) have been efficacious in lowering plasma glucose and enhancing glucose-stimulated insulin secretion, but they carry a risk of overly activating GK and causing hypoglycemia. The theoretical models proposed to date attribute the positive cooperativity of GK to the existence of distinct protein conformations that interconvert slowly and exhibit different affinities for glucose. Here we report the respective crystal structures of the catalytic complex of GK and of a GK-glucose complex in a wide open conformation. To assess conformations of GK in solution, we also carried out small angle x-ray scattering experiments. The results showed that glucose dose-dependently converts GK from an apo conformation to an active open conformation. Compared with wild type GK, activating mutants required notably lower concentrations of glucose to be converted to the active open conformation. GKAs decreased the level of glucose required for GK activation, and different compounds demonstrated distinct activation profiles. These results lead us to propose a modified mnemonic model to explain cooperativity in GK. Our findings may offer new approaches for designing GKAs with reduced hypoglycemic risk.