Catalytic activation of human glucokinase by substrate binding – residue contacts involved in the binding of D-glucose to the super-open form and conformational transitions ˚ Janne Molnes1,2,3, Lise Bjørkhaug1,2, Oddmund Søvik1, Pal R Njølstad1,4 and Torgeir Flatmark3 Department of Clinical Medicine, University of Bergen, Norway Center for Medical Genetics and Molecular Medicine, Haukeland University Hospital, Bergen, Norway Department of Biomedicine, University of Bergen, Norway Department of Pediatrics, Haukeland University Hospital, Bergen, Norway Keywords catalytic activation; D-glucose binding; glucokinase; hysteresis; intrinsic tryptophan fluorescence Correspondence T Flatmark, Department of Biomedicine, University of Bergen, N-5009 Bergen, Norway Fax: +47 55586360 Tel: +47 55586428 E-mail: torgeir.flatmark@biomed.uib.no (Received 19 December 2007, revised March 2008, accepted 10 March 2008) doi:10.1111/j.1742-4658.2008.06391.x a-d-Glucose activates glucokinase (EC 2.7.1.1) on its binding to the active site by inducing a global hysteretic conformational change Using intrinsic tryptophan fluorescence as a probe on the a-d-glucose induced conformational changes in the pancreatic isoform of human glucokinase, key residues involved in the process were identified by site-directed mutagenesis Single-site W fi F mutations enabled the assignment of the fluorescence enhancement (DF ⁄ F0) mainly to W99 and W167 in flexible loop structures, but the biphasic time course of DF ⁄ F0 is variably influenced by all tryptophan residues The human glucokinase–a-d-glucose association (Kd = 4.8 ± 0.1 mm at 25 °C) is driven by a favourable entropy change (DS = 150 ± 10 JỈmol)1ỈK)1) Although X-ray crystallographic studies have revealed the a-d-glucose binding residues in the closed state, the contact residues that make essential contributions to its binding to the superopen conformation remain unidentified In the present study, we combined functional mutagenesis with structural dynamic analyses to identify residue contacts involved in the initial binding of a-d-glucose and conformational transitions The mutations N204A, D205A or E256A ⁄ K in the L-domain resulted in enzyme forms that did not bind a-d-glucose at 200 mm and were essentially catalytically inactive Our data support a molecular dynamic model in which a concerted binding of a-d-glucose to N204, N231 and E256 in the super-open conformation induces local torsional stresses at N204 ⁄ D205 propagating towards a closed conformation, involving structural changes in the highly flexible interdomain connecting region II (R192N204), helix (V181-R191), helix (D205-Y215) and the C-terminal helix 17 (R447-K460) Glucokinase (GK), ATP : d-hexose 6-phosphotransferase (EC 2.7.1.1), catalyses the phosphorylation of a-dglucose (Glc) to form glucose 6-phosphate, using MgATP2) as the phosphoryl donor It is a key regula- tory enzyme in the pancreatic b-cell [isoform of human glucokinase (hGK)] [1] and plays a crucial role in the regulation of insulin secretion, and has been termed the glucose sensor of the b-cell [2] GK is also Abbreviations CR, connecting region; Glc, a-D-glucose; GNM, Gaussian network model; GST, glutathione S-transferase; hGK, human glucokinase; ITF, intrinsic tryptophan fluorescence; MH, a-D-mannoheptulose; MODY2, maturity-onset diabetes of the young type 2; nH, Hill coefficient; PDB, protein databank FEBS Journal 275 (2008) 2467–2481 ª 2008 The Authors Journal compilation ª 2008 FEBS 2467 Activation of glucokinase by D-glucose binding J Molnes et al expressed in the liver (hGK isoforms and 3) [3] and in the central nervous system (hGK isoform 1) [4], where the enzyme has a similar important function in glucose metabolism In humans, a number of naturally occurring mutations in the GK gene (GCK) have been detected in patients suffering from familial, mild fasting hyperglycemia (maturity-onset diabetes of the young type 2; MODY2), persistent hyperinsulinemic hypoglycemia of infancy and permanent neonatal diabetes mellitus [5–7] Although GK is a monomeric enzyme, it shows nonhyperbolic (sigmoidal) dependence on Glc concentration in steady-state enzyme kinetics [8,9] However, the equilibrium binding of Glc alone is characterized by a hyperbolic binding isotherm, as first determined by intrinsic tryptophan fluorescence (ITF) spectroscopy of rat liver glucokinase [10] This enzyme [10] and the recombinant human enzyme [11] are both activated in vitro by incubation with Glc, and the process has been described as a reversible transition from an inactive, low affinity state to a high activity, higher affinity state [10,11] Crystal structure analyses of the unliganded and Glc-bound hGK [12] have confirmed the biochemical and biophysical studies by demonstrating that the binding of Glc at the active site indeed induces a large-scale domain movement that closes the active site cleft and creates the stereochemical environment for binding of the cosubstrate (MgATP2)) and thus catalysis Moreover, the maximal activation of rat liver glucokinase by Glc and the related overall conformational transition, as followed in real-time by ITF spectroscopy, was shown to be a relatively slow process [10] characteristic of a hysteretic enzyme [13] Although X-ray crystallographic studies have revealed the structures of the unliganded (super-open) and Glc-bound (fully-closed) states of hGK [12], the residue contacts that make essential contributions to the binding of Glc to the super-open conformation have not been identified The characterization of these residues is important for our understanding of how substrate binding is coupled to the global conformational transition and catalytic activation In the present study, recombinant wild-type hGK and selected mutant forms were isolated aiming: (a) to examine the contribution of its three tryptophan residues (Fig 1A) to the multiphasic fluorescence enhancement induced by Glc binding; (b) to identify the active site residues involved in the binding of Glc to the super-open state (Fig 1C) of this two domain [large (L) and small (S)] enzyme, and thus the site of initiation of the global conformational transition; and (c) to gain some insight into how the local torsional stresses at the contact residues in the super-open state propagate through the structure towards cleft closure and a 2468 A W167 W99 Glc W257 CompA T168 B E290 K169 E256 N231 D205 N204 C N204 D205 E256 N231 E290 Fig (A) Localization of tryptophan residues in the 3D structure of the fully-closed state of wild-type hGK with bound D-glucose (Glc) and the allosteric activator compound A (PDB identity: 1v4s) (B) The Glc contact residues in the substrate-bound state (PDB identity: 1v4s) All residues were individually mutated (C) The spacial proximity of active site residues in the L-domain and connecting region II which are potentially involved in the binding of D-glucose in the super-open state (PDB identity: 1v4t) The structural images were generated using PYMOL, version 0.99 catalytically competent conformation To explore these aspects, we used a combined approach of molecular dynamics studies by real-time ITF spectroscopy, structural dynamic analyses and functional mutagenesis Our findings provide new insight into the catalytic activation of hGK by substrate binding that will be valuable in studies of human diseases associated with mutations in the GCK gene, notably in some mutations in which the molecular mechanism is not yet understood FEBS Journal 275 (2008) 2467–2481 ª 2008 The Authors Journal compilation ª 2008 FEBS Activation of glucokinase by D-glucose binding J Molnes et al 26.3° and )0.2°, respectively It should be noted that residues 157–179, unassigned in the electron density map of the super-open structure (1v4t), were ‘repaired’ by the molmovdb algorithm Results Tryptophan residues in wild-type hGK The crystal structures of hGK [12] have identified the positions and the interactions of its three tryptophans in the absence (super-open state) and in the presence (fullyclosed state) of Glc and 2-amino-4-fluoro-5-(1-methyl1H-imidazol-2-ylsulfanyl)-N-thiazol-2-yl-benzamide, a synthetic allosteric activator termed compound A (Fig 1A) Based on the coordinates of the two structures [protein databank (PDB) identity: 1v4t and 1v4s], molecular motion analyses (http://molmovdb.mbb yale.edu/cgi-bin/morph.cgi?ID=496337-23316) revealed a change in the backbone dihedral torsion angle (Du + Dw) for W99, W167 and W257 to be 110.5°, A 100 Steady-state kinetics of wild-type hGK and the W fi F mutant forms As previously reported [14,15], the wild-type hGK and wild-type glutathione S-transferase (GST)-hGK demonstrated the same steady-state kinetic parameters as well as the Kd value for Glc in the ITF binding assay (Fig 2C), and the GST fusion proteins were therefore mostly used in the kinetic analyses of mutant proteins The wild-type GST-hGK revealed a positive kinetic cooperativity with Glc [Hill coefficient (nH) = 1.7 C 80 60 340.5 nm 40 20 Feq / Fo (normalized) Fluorescence intensity 338.6 nm 1.0 [r = 0.99] 0.8 0.6 0.4 0.2 0.0 320 340 360 380 400 420 440 20 340.3 nm D 5.8 140 120 [r = 0.96] 5.6 341.0 nm 5.4 100 –ln Kd Glc Fluorescence intensity B 160 60 40 [Glc] (mM) Wavelength (nm) 80 60 40 5.2 5.0 4.8 4.6 20 4.4 4.2 320 340 360 380 400 420 440 3.3 Wavelength (nm) 3.4 3.5 3.6 x103 (K–1) T Fig Effect of D-glucose on the equilibrium fluorescence of wild-type hGK and wild-type GST-hGK (A) and (B) The fluorescence spectra of the enzyme (25 °C) in the absence (solid line) and presence (short dashes) of 200 mM Glc for wild-type hGK (A) and wild-type GST-hGK (B) (C) The Glc binding isotherm at 25 °C was obtained by monitoring the enhancement in ITF of wild-type GST-hGK (s) and wild-type hGK ( ) by increasing concentrations of Glc The solid lines represent the fit of the data to two hyperbola as obtained by nonlinear regression analyses, giving Kd values of 4.8 ± 0.1 mM and 4.9 ± 0.1 mM for wild-type GST-hGK and wild-type hGK, respectively For both plots, the value at 60 mM Glc is normalized to (D) van’t Hoff analysis of the temperature dependence of the apparent dissociation constant (Kd) for the hGKGlc interaction measured at 7, 12, 17.5, 22.5, 27.5 and 31.5 °C A least-square linear fit (r2 = 0.96) yields a DHvan’t Hoff of 32 ± kJỈmol)1 and a DS of 150 ± 10 JỈmol)1ỈK)1, indicating that the interaction is driven by a favourable entropy change The measurements were performed using 0.03 mgỈmL)1 wild-type GST-hGK in 20 mM Hepes, 100 mM NaCl and mM dithiothreitol (pH 7.0) The excitation wavelength was 295 nm and excitation and emission slits were and nm, respectively (A, B), or and nm, respectively (C, D) FEBS Journal 275 (2008) 2467–2481 ª 2008 The Authors Journal compilation ª 2008 FEBS 2469 Activation of glucokinase by D-glucose binding J Molnes et al Table Steady-state kinetic parameters of wild-type GST-hGK and its Trp mutant forms GST-hGK Vmaxa (nmolỈmg)1Ỉs)1) kcat (s)1) [S]0.5a (mM) kcat ⁄ [S]0.5 (mM)1Ỉs)1) n Ha Wild-type W99F W167F W257F 795 616 229 554 60.4 46.8 17.4 42.1 8.4 6.2 55.2 11.4 7.2 7.5 0.3 3.7 1.71 1.75 1.12 1.66 a ± ± ± ± 12 10 14 ± ± ± ± 0.9 0.6 0.8 1.1 ± ± ± ± 0.2 0.2 5.3 0.5 ± ± ± ± 0.06 0.08 0.08 0.10 Based on nonlinear regression and the Hill equation ± 0.1] with a substrate concentration yielding halfmaximum saturation ([S]0.5) of 8.4 ± 0.2 mm, whereas all three W fi F mutants demonstrated a reduced catalytic activity (Table 1) W167F-hGK showed a pronounced reduction in ‘catalytic efficiency’ (24fold), with an approximate three-fold reduction in Vmax and a six-fold increase in the [S]0.5 value for Glc, and the Hill coefficient was reduced to nH = 1.12 ± 0.08 The W257F mutant also revealed a slightly reduced affinity for Glc, whereas W99F showed a small increase in both affinity and ‘catalytic efficiency’ A normal positive kinetic cooperativity was observed for the W99F and W257F mutant forms Correlation of tryptophan environments with fluorescence properties The static solvent accessibility of W99, W167 and W257 in the super-open ⁄ fully-closed state was calculated [16] as 27 ⁄ 45, X ⁄ 4.6 and 0.8 ⁄ 0.0%, respectively W167 and W257 are ‘buried’ tryptophans, whereas W99 is a surface residue with a high degree of exposure to aqueous solvent in both states, notably in the Glc-bound state Note that no number exists for W167 in the super-open state because residues 157–179 are unassigned in the electron density map [12] Figure 3A shows the fluorescence emission spectrum (kex = 295 nm) of wild-type hGK at pH 7.0 with kmax $ 340.5 nm (kmax $ 341 nm for wild-type GSThGK; Fig 2B), consistent with the solvent accessibility of the three tryptophans On denaturing with m guanidium hydrochloride, a red shift was observed (kmax $ 357 nm), close to the spectrum for free tryptophan (data not shown) On increasing the temperature from °C to 37 °C, an approximately 25% decrease in the fluorescence intensity at kmax and an approximately 5.1 nm red shift in the kmax were observed (Fig 3B) These changes suggest a complex effect of temperature on the conformational substates of the apo enzyme, presumably with a more solvent-exposed W99 at the higher temperature Moreover, in rapid mixing experi2470 ments a temperature change from °C to 39 °C resulted in a time dependent quenching of the fluorescence within a time scale of approximately (Fig 3C) A semi-log plot (Fig 3C, inset) revealed a biphasic time course with a relatively fast phase (t1 ⁄ $ 11 s) and a slow phase (t1 ⁄ $ 64 s) Effect of D-glucose on the equilibrium fluorescence of wild-type hGK and its W fi F mutant forms From the equilibrium fluorescence spectra of wildtype hGK (Fig 2A), it is seen that, upon the addition of 200 mm Glc, fluorescence (DFeq ⁄ F0) increases by approximately 60%, with 1.9 nm blue shift in kmax A similar effect was seen with the wild-type GSThGK fusion protein (Fig 2B) The increase in fluorescence units was comparable without (DFeq ) F0 $ 33) and with (DFeq ) F0 $ 30) fusion partner, considering the experimental error in determining the absorption coefficients at 280 nm for the two proteins No effect of Glc was observed on the fluorescence spectrum of the isolated GST protein (data not shown) The protein with and without fusion partner also shows the same time-dependence of the fluorescence enhancement (see below) and revealed identical hyperbolic binding isotherms for Glc with Kd values of 4.8 ± 0.1 and 4.9 ± 0.1 mm, respectively, at 25 °C (Fig 2C) These data demonstrate that the fusion partner at the N-terminal does not perturb the substrate-induced conformational changes of hGK, and the GST fusion proteins were therefore used alternatively in the studies of mutant proteins due to their potentially higher in vitro stability To better understand the driving force of the hGK–Glc interaction, the temperature dependence was determined in the range 7–32 °C (Fig 2D) A least-square linear fit (r2 = 0.96) yields a DHvan’t Hoff of 32 ± kJỈmol)1 from the slope and a DS of 150 ± 10 JỈmol)1ỈK)1 from the y-axis intercept Thus, the favourable DS overcomes the unfavourable DH and drives the association between hGK and Glc FEBS Journal 275 (2008) 2467–2481 ª 2008 The Authors Journal compilation ª 2008 FEBS B 160 A 340.5 nm Fluorescence intensity Fluorescence intensity 60 50 40 30 20 10 140 338.6 nm °C 120 343.7 nm 100 37 °C 80 150 140 130 120 110 10 15 20 25 30 35 40 Temp ( ° C) 60 40 20 0 350 320 340 360 380 400 420 440 Wavelength (nm) C 400 450 500 Wavelength (nm) 2.2 F t - F eq Ft= - Feq x 100 135 130 125 log Fluorescence intensity Fluorescence intensity Activation of glucokinase by D-glucose binding J Molnes et al 2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 120 50 100 150 Time (s) 115 110 100 200 300 400 Time (s) Fig Steady-state fluorescence of hGK (A) The spectrum of the isolated wild-type hGK (0.03 mgỈmL)1) obtained in the unliganded superopen state at 25 °C (B) The effect of temperature (7 °C to 37 °C) on the fluorescence intensity of wild-type hGK (0.063 mgỈmL)1) The observed red-shift in kmax with increasing temperature is emphasized The graph (inset) shows the temperature dependence of the fluorescence intensity at kmax (C) A typical ‘temperature-jump’ experiment (measured over a period), in which the isolated wild-type hGK enzyme (0.063 mgỈmL)1) stored in buffer at approximately °C was rapidly ($ s) mixed in the cuvette at 39 °C, demonstrating the expected decrease in fluorescence intensity at k = 340 nm, with an end point at approximately The semi-log plot (inset) shows a biphasic time course with t1 ⁄ $ 11 s and t1 ⁄ $ 64 s Ft = is the fluorescence intensity measured immediately after mixing of the enzyme (< s after addition), and Feq is the equilibrium fluorescence intensity The fluorescence was read every 0.1 s, and Ft is the average fluorescence intensity of 20 data points (2 s) (first phase) or 100 or 200 data points (10 s or 20 s, respectively) (second phase) The data were analysed by linear-regression analysis using SIGMAPLOT TECHNICAL GRAPHING Software The excitation wavelength was 295 nm, and excitation and emission slits were and nm in (A, B) or and nm in (C), respectively To simultaneously demonstrate the effect of Glc on both the fluorescence enhancement and the spectral shifts, the +Glc ⁄ )Glc fluorescence difference spectra were recorded for wild-type and W fi F mutant forms The difference spectrum of wild-type GST-hGK (DFeq ⁄ F0 $ 27%) revealed a kmax $ 334 nm (Fig 4A) compatible with an additive contribution of the three tryptophans to the Glc-induced fluorescence enhancement At identical protein concentrations, all the W fi F mutant forms resulted in a decreased Glc-induced fluorescence enhancement, being most pronounced for W99 (DFeq ⁄ F0 $ 10%) (Fig 4B) and W167 (DFeq ⁄ F0 $ 6%) (Fig 4C), whereas a DFeq ⁄ F0 value of approximately 19% was observed for the W257F mutant form (Fig 4D) The W167F and W257F mutants revealed only an approximate nm shift in kmax in the +Glc ⁄ )Glc difference spectra compared to wild-type GST-hGK, whereas the W99F mutant demonstrated an approximately 11 nm blue shift, as expected from the low solvent accessibility of the remaining W167 and W257 residues For wild-type GST-hGK and the two ‘buried’ tryptophan mutants (W167F and W257F), a close correlation (r2 = 0.99) was observed between the DFeq ⁄ F0 value and the catalytic activity at 200 mm Glc (Fig and supplementary Table S1) This correlation is presumably related to a variable perturbation of the overall structural dynamics in the W167F and W257F mutant forms that affects the time-dependent Glc-induced conformational change (supplementary Fig S1) and catalytic activation to the same extent This is in contrast to the W99F mutant form because W99 is more solvent FEBS Journal 275 (2008) 2467–2481 ª 2008 The Authors Journal compilation ª 2008 FEBS 2471 Activation of glucokinase by D-glucose binding 30 B 334.3 nm 20 10 20 323.4 nm 10 ∼ 332 nm 320 340 360 380 400 420 Wavelength (nm) Fluorescence difference 10 W99F W167F 0.2 0.4 0.6 0.8 1.0 1.2 Relative catalytic activity 30 Fig Correlation between the catalytic activity and the D-glucose induced fluorescence enhancement of wild-type GST-hGK and W fi F mutant forms Shown are the values for catalytic activity at 200 mM Glc (relative to wild-type hGK) and the corresponding values for fluorescence intensity (Table S1) A linear correlation of r2 = 0.99 was obtained for the wild-type hGK, W167 and W257 mutant forms Graphic points including error bars represent the mean ± SD of three or four measurements 336.4 nm 20 10 320 340 360 380 400 420 Wavelength (nm) Fig Effect of D-glucose on the fluorescence of W fi F mutant forms The Glc-induced fluorescence changes of the GST fusion proteins (0.03 mgỈmL)1) upon addition of 200 mM Glc are shown as fluorescence difference spectra Each spectrum was obtained by subtracting the signal averaged spectra obtained in the absence of Glc from the spectra obtained in the presence of Glc (A) Wild-type GST-hGK; (B) W99F GST-hGK; (C) W167F GST-hGK and (D) W257F GST-hGK All spectra were obtained at 25 °C with an excitation wavelength of 295 nm and excitation and emission slit widths of and nm, respectively exposed in a highly flexible surface loop The far-UV CD spectrum of wild-type hGK revealed negative bands at 208.5 and 222 nm (data not shown) characteristic of a protein predominated by a-helical secondary structure, with an apparent a-helical content of approximately 31% W167F-hGK revealed a similar CD spectrum, but with an estimated slightly reduced a-helical content The thermal denaturation profiles of the two proteins, as measured at 222 nm in the presence of 50 mm Glc, gave Tm values of 44.2 °C and 42.4 °C for wild-type hGK and W167F-hGK, respectively These data demonstrate that the secondary structure and conformational stability of the W167F mutant is relatively well preserved, and support the conclusion that the functional effects of the W fi F mutation are presumably mainly related to a structural perturbation due to its localization next to T168 and K169 whose side-chains normally form hydrogen bond interactions with Glc in the fully-closed conformation 2472 15 Wavelength (nm) D 20 W257F 320 340 360 380 400 420 Wavelength (nm) 30 20 10 C WT 25 320 340 360 380 400 420 Fluorescence difference [r = 0.99] 30 ΔFeq / Fo 30 Fluorescence difference Fluorescence difference A J Molnes et al D-glucose-induced conformational dynamics The time course of the fluorescence enhancement induced by Glc was followed on a second-to-minute time scale As shown in Fig 6, a rapid initial phase (0–5 s) represented approximately 80% of the total increase in fluorescence of wild-type hGK (Fig 6A) and wild-type GST-hGK (Fig 6B), and includes the two phases observed by transient kinetics [11], but the equilibrium level (DFeq ⁄ F0) was not reached until approximately at 25 °C The data in Fig 6C refer to the total fluorescence change, DFeq ⁄ F0, (black bars) or the amplitude of the fast phase, DFinitial ⁄ F0 (gray bars) or the slow phase, DFslow ⁄ F0, (open bars), all relative to the baseline value F0 A biphasic time course was also observed for the W99F and W257F mutant forms (supplementary Fig S1B,D) although the total amplitude at equilibrium and the relative proportion of the two phases varied (Fig 6C), and the time required to reach the equilibrium value increased By contrast, in the W167F mutant form the rapid phase dominated, with a scarcely detectable slow phase, and the overall amplitude was markedly reduced (Fig 6C and supplementary Fig S1C) This may be related to the loss of kinetic cooperativity of Glc binding (mH = 1.12 ± 0.08; Table 1) a-d-Mannoheptulose (MH) is a nonmetabolized competitive inhibitor of GK This Glc analogue has been proposed to bind at the catalytic site in the closed conformation of GK [17,18] with a 50% inhibition at FEBS Journal 275 (2008) 2467–2481 ª 2008 The Authors Journal compilation ª 2008 FEBS Activation of glucokinase by D-glucose binding J Molnes et al Fluorescence intensity A Functional mutation analysis of Glc contact residues at the active site 100 90 80 70 60 50 100 200 300 200 300 Time (s) Fluorescence intensity B 140 130 120 110 100 100 Time (s) C 30 ΔFluorescence 25 20 15 10 WT W99F W167F W257F Fig The time-dependent D-glucose-induced fluorescence enhancement of wild-type hGK, wild-type GST-hGK and its W fi F mutant forms (A, B) The time course for the Glc-induced fluorescence enhancement of wild-type hGK (A) and wild-type GST-hGK (B) as measured on a time scale of 0–6 min, at 200 mM Glc (C) A comparison of the time-dependent fluorescence enhancement in wild-type GST-hGK and its W fi F mutant forms Shown are the values listed in supplementary Table S1 for the total change in ITF upon addition of 200 mM Glc, measured as DFeq ⁄ F0 (black bars) or the amplitude of the fast phase DFinitial ⁄ F0 (i.e 0–5 s; gray bars) and slow phase DFslow ⁄ F0 (i.e s to min; open bars) The change in ITF was followed at 25 °C with an excitation wavelength of 295 nm and excitation and emission slit widths of and nm, respectively Each column represents the mean ± SD of three measurements approximately mm [19] From supplementary Fig S2A, it is seen that MH binds to the super-open conformation and induces an equilibrium enhancement of hGK fluorescence similar to Glc and with a similar biphasic time dependency From the hyperbolic binding isotherm (supplementary Fig S2B), a Kd of 8.0 ± 0.7 mm was calculated at 25 °C The 3D structure of the closed state of hGK (PDB identity: 1v4s) has revealed that Glc is hydrogen bonded to amino acids in the L-domain (residues N204, D205, N231, E256 and E290) and the S-domain (residues T168 and K169) (Fig 1B) To identify the contact residues involved in the initial binding of Glc to the super-open state (Fig 1C) of this two domain hinge-bending enzyme, all the actual residues were individually mutated (supplementary Table S2) The mutant forms were expressed as GST-fusion proteins and subjected to steady-state enzyme kinetics and Glc-induced fluorescence enhancement analysis (Table 2) The main results of this screen (Table 2) are alternatively shown in supplementary Fig S3, including the ‘catalytic efficiency’ (kcat ⁄ [S]0.5) (black bars) and the fluorescence enhancement at 200 mm Glc, (DFeq ⁄ F0)max (gray bars) The mutations in the L-domain (N204A, D205A and E256A ⁄ K) resulted in enzyme forms that did not give any fluorescence enhancement by Glc and they were essentially catalytically inactive at a Glc concentration of 200 mm N231A gave a DFeq ⁄ F0 response of approximately 6% versus wild-type and no measureable activity By contrast, the mutations in the S-domain (T168G and K169N) experienced a variable partial loss ($ 20– 40%) of Glc-induced fluorescence enhancement, with an increased Kd value and reduced catalytic activity (Table 2) The titration curves for the mutants T168G, K169N and Q287V all revealed clear hyperbolic binding isotherms for Glc (r2 = 0.99) (data not shown) For the mutant N231A, the accuracy of the experiments was hindered by the low fluorescence response to Glc (DFeq ⁄ F0 at 200 mm Glc $ 6%), but the data were fitted to a hyperbolic binding curve (r2 = 0.91) (data not shown) Structural dynamic analyses 3D structural analyses of the Glc-induced conformational changes [12] revealed that the enzyme is a very dynamic structure with a high conformational flexibility The crystallographic B factor values for Ca carbons (Fig 7A), demonstrating the freedom and restriction for various sites, revealed low values (£ 30 A2) for the Glc-interacting residues in the unliganded state, except for T168 and K169 The conformational fluctuations, computed by the Gaussian network model (GNM) [20,21], revealed similar sites (minima) of low translation mobility compatible with N204, D205, N231 and E256 (Fig 7B) as potential FEBS Journal 275 (2008) 2467–2481 ª 2008 The Authors Journal compilation ª 2008 FEBS 2473 2474 9.4 26.7 0.2 1.01 14.7 17.8 100 8.4 ± 0.2 7.2 1.71 ± 0.06 25.7 ± 0.1 4.8 ± 0.1 ± 0.05 ± 0.1 ± 0.3 ± 1.5 75 ± 795 ± 12 8.9 976 ± 89 5.5 · 10)3 1.31 ± 0.05 NM NM 28.2 ± 2.1 6.7 · 10)4 0.98 ± 0.08 19.9 ± 0.2 47.9 ± 1.4 71 0.25 ± 0.01 0.03 N204A K169N NM NM NM NM NM NM NMa D205A NMb NMb NMb 1.5 ± 0.1 21.9 ± 7.1 0.03 0.2 N231A NM NM NM NM NM NM NMc E256K 57.9 35.1 1.0 1.22 21.5 23.3 1625 ± 359 9.8 · 10)4 1.46 ± 0.10 NM NM ± 0.03 ± 0.2 ± 0.9 ± 0.7 460 ± Q287V 2.6 21 E256A 829 ± 196 0.03 1.03 ± 0.06 11.7 ± 0.4 395 ± 22 45.9 365 ± 47 E290A Fluctuations B-factor (A2) Catalytic activity was not detectable at 200 times the enzyme concentration used for wild-type hGK b The mutant form had no catalytic activity at physiological [Glc] We were unable to estimate nH and the [S]0.5 for Glc c Catalytic activity was nondetectable at 100 times the enzyme concentration used for wild-type hGK a Catalytic activity (nmolỈmg)1Ỉs)1) Relative catalytic activity (%) [S]0.5 Glc (mM) kcat ⁄ [S]0.5 (mM)1Ỉs)1) Hill coefficient (nH) (DFeq ⁄ F0)max Kd Glc (mM) T168G Wild-type Table Steady-state kinetics and fluorescence properties of wild-type GST-hGK and its active site mutant forms Each number was obtained from measurements at 12–15 different glucose concentrations The mutations N204A, D205A, N231A and E256A ⁄ K resulted in enzyme forms that not bind Glc at all at physiological concentrations of Glc and they are essentially catalytically inactive Only in the range 200–1600 mM was measurable activity observed for N204A, N231A and E256A The catalytic activity presented for these mutants is the activity measured at a Glc concentration of 200 mM NM, not measurable Activation of glucokinase by D-glucose binding J Molnes et al A 100 80 0.000 T168 K169 E256 E290 60 100 0.012 0.010 100 Q287 N204 D205 N231 40 20 200 Residues 0.008 0.006 200 300 T168 K169 N204 D205 300 400 B 0.014 E290 Q287 N231 E256 0.004 0.002 Residues 400 Fig Crystallographic B-factor values for Ca carbons (A) and mobilities in the global modes (B) for the unliganded state of wildtype hGK (PDB identity: 1v4t) The residue fluctuations (B) were predicted by the GNM [20,21], and the profile represents the slowest frequency mode As indicated, the residues 157–179 of the S-domain are unassigned in the electron density map Table Changes in backbone and side-chain dihedral angles of Glc contact residues in wild-type hGK on binding Glc Values are calculated from the coordinates of the super-open (PDB identity: 1v4t) and fully-closed (PDB identity: 1v4s) state Residue (Du + Dw) Dv1 Dv2 N204 D205 N231 E256 )2.1 0.3 5.3 )4.1 15.4 177.7 0.4 33.3 178.9 16.5 8.6 32.0 ligand binding sites The binding of Glc changes not only the tertiary structure (large scale domain motion), but also the secondary structure and side-chain positioning ⁄ interactions Thus, 17 helices were identified in the unliganded super-open state (PDB identity: 1v4t) versus 19 helices in the Glc and allosteric activatorbound closed state (PDB identity: 1v4s) The changes in the backbone and side-chain dihedral angles for the Glc contact residues are shown in Table FEBS Journal 275 (2008) 2467–2481 ª 2008 The Authors Journal compilation ª 2008 FEBS J Molnes et al Activation of glucokinase by D-glucose binding It should be noted that the GK activator (compound A; Fig 1A) binds to the binary hGKỈGlc complex, but it is not known in what way its binding perturbs the structure [12] Discussion The multiphasic global conformational transition and the kinetic cooperativity of D-glucose binding Based on enzyme kinetic studies [8,9], crystal structure analyses [12] and real-time ITF spectroscopy [10,11,22], there appears to be broad agreement that the catalytic activation of monomeric GK by its substrate Glc can be presented by the equation: k1 k2 kÀ1 kÀ2 GK ỵ Glc $ GK Glc $ GK Glc ð1AÞ where GK represents the ligand-free, inactive state of the enzyme, GK Ỉ Glc its binary low-activity (lowaffinity) enzyme–substrate complex and GK* Ỉ Glc the binary complex of the high-activity (higher affinity) state of the enzyme in which a relatively slow conformational change (isomerization) has occurred, characteristic of a hysteretic enzyme [13] In transient kinetic analyses of the Glc-induced enhancement of ITF with wild-type hGK [11] and seven activating mutations [22], a biphasic time course was observed suggesting two kinetically distinguishable events within the time scale of 0–5 s The observed rate constant for the first phase, kobs1, was linearly dependent on the Glc concentration, whereas the second-phase rate constant, kobs2, exhibited a hyperbolic dependence on the substrate concentration The amplitude of the first and second phase represented approximately 25% and 75%, respectively, of the total fluorescence enhancement of wild-type hGK [11] Based on these analyses, it was concluded that the positive cooperativity of GK observed in enzyme kinetics [8,9] is a kinetic behavior that is mediated by the Glc-induced conformational change with intermediate stable states of different affinity for Glc In previous transient kinetic analysis [11], the time scale was 0–5 s, whereas, in the present study (Fig 6), the equilibrium enhancement (DFeq ⁄ F0) of ITF is not reached until approximately at 25 °C in wildtype hGK (and wild-type GST-hGK) and found to be very temperature dependent (data not shown) This suggests that more than two discernible consecutive steps (Eqn 1A) may accompany Glc recognition and binding at the active site, a conclusion that is further supported by the molecular dynamics and targeted molecular dynamics simulations on the enzyme in its transition from the fully-closed to the super-open state [23] The simulations indicate that the overall conformational transition includes three likely stable intermediate states with variable degrees of cleft opening Our results provide an additional framework to understand the Glc-induced enhancement in ITF of wild-type hGK First, the W fi F mutation analyses reveal that all the three tryptophans contribute to the overall enhancement of ITF induced by Glc, with major contributions of W99 and W167 (Figs and and supplementary Table S1), both located in highly flexible loop structures W99, located in one of the regions connecting the L- and S-domains, undergoes a large change in the backbone conformation [(Du + Dw) of $ 110°] upon Glc binding, whereas the corresponding change in the microenvironment is more uncertain for the buried W167 (residues 157–179 not assigned in the super-open state) Second, the hGK– Glc association (Kd = 4.8 ± 0.1 mm at 25 °C) is driven by a favourable entropy change (DS = 150 ± 10 JỈmol)1ỈK)1), which overcomes an unfavourable enthalpy change (DHvan’t Hoff = 32 ± kJỈmol)1) The relatively large positive DS is in keeping with that an increase in protein dynamics plays a dominant role in the interaction, with large scale domain movement and cleft closure (desolvation) as well as changes in peptide backbone conformation ⁄ side-chain rotameric states [12] Finally, the temperature induced ($ °C to 39 °C) reversible quenching of ITF (Fig 3C) is consistent with a slow conformational isomerization, and the biphasic time course (t1 ⁄ $ 11 s and t1 ⁄ $ 64 s) suggests the presence of a relatively stable intermediate in the transition This reversible isomerization (‘thermal hysteresis’) is reminiscent of the Glc-induced conformational isomerization, and supports the existence of an equilibrium between conformational substates in the apo enzyme Interestingly, recent pre-steady-state analyses of Glc binding to wild-type hGK [24] have provided evidence that the substrate-free enzyme in solution is in a preexisting equilibrium between at least two conformers (i.e super-open and closed) which differ in their affinity for Glc as presented by the equation: GK ỵ Glc $ GK Glc $ GK Glc l GK ỵ Glc $ GKÃ Á Glc ð1BÞ where the binding of Glc shifts the equilibrium towards the high activity (higher affinity) closed state of the enzyme (GK*) In the present study, the time course and equilibrium (DFeq ⁄ DF0) fluorescent enhancement induced by Glc were studied in the absence of MgATP2) because FEBS Journal 275 (2008) 2467–2481 ª 2008 The Authors Journal compilation ª 2008 FEBS 2475 Activation of glucokinase by D-glucose binding J Molnes et al such data are not complicated by turnover conditions with the formation of glucose 6-phosphate The possibility, therefore, arises that the ITF responses and the related structural conformational changes measured for the GKỈ Glc binary complex may be different in the GKỈ Glc Ỉ MgATP ternary complex The question was recently addressed by Kim et al [24] using a nonhydrolyzable ATP analogue (PNP-AMP) Their transient kinetic analyses suggested that PNP-AMP may change the equilibrium between the two proposed GK conformers (Eqn 1B), but the accuracy of the experiments was hindered by the low signal amplitude [24] Therefore, they also studied the equilibrium binding of PNP-AMP to the enzyme, and reported a relatively large decrease in fluorescence (DFeq ⁄ F0) which was interpreted as a nucleotide induced conformational change However, because no corrections were made for the large inner-filter effect due to the significant absorbance of the nucleotide at the selected excitation wavelength of 285 nm, further fluorescence analyses in which proper corrections are made for the inner-filter effect are required before any conclusions can be drawn Additionally, Heredia et al [11] have performed differential scanning calorimetry of wild-type hGK and concluded that 10 mm MgATP2), in contrast to 100 mm Glc, did not have any significant effect on the Cpexc (kcalỈmol)1Ỉ°C)1) and thermal midpoint transition temperature, further supporting the conclusion that more studies are required to settle the issue of a possible MgATP2) induced conformational change in GK Residues involved in the binding of D-glucose to the super-open conformation The 3D structure has revealed that GK is a typical two-domain enzyme and, in the unliganded state, the L- and S-domains are far apart and bisected by a wide open, solvent-accessible cleft [12] In the Glc-bound fully-closed state, the two domains are in close proximity, and the desolvated ligand is engulfed in the cleft and held in place by extensive hydrogen-bonding interactions with residues in the L-domain (residues N204, D205, N231, E256 and E290) and the S-domain (residues T168 and K169) (Fig 1B) In the super-open state, however, these contact residues are too far apart to simultaneously interact with Glc Those residues involved in the first binding of Glc to the super-open conformation and the subdomain in which the global conformational transition is initiated have not yet been experimentally identified Our point mutation analysis provides experimental evidence that Glc binds first to residues in the 2476 L-domain and subsequently (after closure) to residues in the S-domain The mutations N204A, D205A and E256A ⁄ K resulted in enzyme forms that did not bind Glc at all as measured by ITF, and they were essentially catalytically inactive (Table and supplementary Fig S3), whereas N231A gave a DFeq ⁄ F0 response of approximately 6% versus wild-type and no measurable activity By contrast, in the mutations of the S-domain (T168G and K169N), Glc induced a significant fluorescence enhancement ($ 60% and 80% versus wild-type), and with reduced affinity (Table and supplementary Fig S3) In the 3D structure of the super-open state, residues 204, 205, 231 and 256 demonstrate spatial proximity, with N204, N231 and E256 in the most favourable positions (Cc-Cc ⁄ Cd distances ˚ in the range of 5.1–5.4 A) and side-chain orientations for a concerted interaction with Glc (Fig 8B) The side-chain of D205, suggested to be the triggering target in Glc binding [12], is, however, in a more unfavourable orientation and forms a salt-bridge with R447 in helix 17 (Fig 9) This stabilizing salt-bridge is broken upon Glc-binding (Fig 8C), and the side-chain of D205 is reorientated [the side-chain dihedral angles change by 117.7° (v1) and 16.5° (v2)] (Table 3), whereas the (Du + Dw) value is changed by only 0.3° Thus, D205 subsequently interacts with Glc In the D205A mutant form, there is no salt bridge, and Ala does not function as a contact residue, offering an explaination for the Glc nonbinding effect of the D205A mutation Local torsional stresses induced by Glc binding and propagation of the conformational transition From the 3D structures of hGK [12], the overall molecular motion induced by Glc binding is characterized by a complex shear ⁄ sliding and hinge type of movements, as previously described for the structurally related hexokinase I [25,26] The core region (middle and outer layers) of the S-domain is rotated by approximately 99° as a rigid body compared to 12° for hexokinase I Whereas three regions, connecting the L- and S-domains, were assigned as hinge regions in hexokinase I [25] no hinge regions were defined for hGK [12] Using the Hinge Master algorithm for prediction of hinge regions in the closed conformation (PDB identity: 1v4s), the highest score was obtained for residues in two of the regions connecting the L- and the S-domains [i.e in connecting region (CR) I (residues 62–73) and in CR II (residues 192–204)] and the crystal structures of the two conformational states demonstrate large changes in the main-chain torsion angles of both regions on Glc binding CR II, which FEBS Journal 275 (2008) 2467–2481 ª 2008 The Authors Journal compilation ª 2008 FEBS Activation of glucokinase by D-glucose binding J Molnes et al 250 A H5 CRII (β8) H17 H6 Dihedral angles (Δϕ + Δ Δψ) 200 V455 150 R447 L451 100 50 K458 ** D205 –50 –100 *N204 and D205 E216 –150 180 190 200 H6 Residue number B C Fig (A) Torsion angle analysis of residues in helix (H5), CR II and helix (H6) illustrating the large changes in main-chain torsion angles which occur at CR II (residues 191–203) upon binding of Glc The position of the Glc contact residues N204 and D205 is indicated by an asterisk (B, C) Demonstrating the related changes in the backbone conformation based on the structural coordinates of the super-open state (PDB identity: 1v4t) and the Glc-bound state (PDB identity: 1v4s), respectively Also note the interhelical contacts between helix (D205-Y215) and the C-terminal helix 17 (R447-K460) in the super-open state The structure illustrates that the inactive form of the enzyme is stabilized by two salt bridges, D205ỈỈỈR447 and E216ỈỈỈK458; not shown are additional pairs of residues (I211 ⁄ Y215 and L451 ⁄ V455) involved in hydrophobic interactions (Fig 9) The structural images were generated using PYMOL, version 0.99 connects helix (S-domain) and helix (L-domain), is of particular interest in the present context due to its connection to the two Glc contact residues N204 and I211 Y215 210 Fig Main interhelical contacts between helix (D205-Y215) and the C-terminal helix 17 (R447-K460) in the super-open state (1v4t) The structure illustrates that the apo enzyme is stabilized by two ˚ salt bridges, D205ỈỈỈR447 (closest distance 3.2 A) and E216ỈỈỈK458 ˚ (closest distance 3.4 A), and pairs of residues (I211 ⁄ Y215 and ˚ L451 ⁄ V455, closest distance £ 3.5 A) involved in hydrophobic interactions Interstices shown using all-atom dot surface The structural image was generated using PYMOL, version 0.99 D205 A torsion angle analysis revealed large changes in the backbone dihedral angles (Du + Dw) of several residues in CR II on Glc binding (Fig 8A), and the related changes in the backbone conformation are shown in Fig 8B,C The short b8 strand is extended by three residues, helix and helix are both shortend by one residue, and the two helices change their relative orientation consistent with hinge-bending motions Thus, the conversion from one backbone conformation to the other of CR II induces large-scale motions in the protein It should be noted that only minor changes in the petide backbone conformation were observed in the regions around the Glc contact residues N231 and E256 as well as in their side-chain rotameric states (Table 3) Stabilizing interactions between helix and helix 17 in the super-open conformation In both the super-open and fully-closed states, helix interacts specifically with the C-terminal helix (helix 17 ⁄ 19), which adopts a different length in the superopen (H17, residues 447–460) and fully-closed (H19, residues 443–461) state, and their relative orientation (the crossing angle increases from 15.8° to 75.6°) and main residue contacts also change noticeably upon Glc binding (Fig 8B,C) The changes in the interhelical interactions point to a major contribution in the dynamic communication between the L- and S-domains and thus in the transition from the super- FEBS Journal 275 (2008) 2467–2481 ª 2008 The Authors Journal compilation ª 2008 FEBS 2477 Activation of glucokinase by D-glucose binding J Molnes et al open (inactive) to the fully-closed (active) conformation The interhelical salt bridges and pairs of residues involved in hydrophobic interactions (Fig 9), as specific structural determinants, stabilize the inactive state of the enzyme On binding Glc, this constrained conformation relaxes and renders the enzyme more active This conclusion is consistent with the targeted molecular dynamics simulations [23] demonstrating that the ‘release’ of the C-terminal helix from the S-domain is a final event in the conformational transition from the fully-closed to the super-open state To date, 11 activating mutations in hGK have been identified [27–29] Two of these (Y214C and Y215A) are localized in helix and three (V455M, A456V and A460R) in helix 17, and some of these missense mutations (e.g Y215A and V455M) perturb the hydrophobic interactions between the two helices that normally stabilize the inactive enzyme conformation The activating mutations result in a variably enhanced affinity for Glc and increased Vmax [27,28] Finally, hGK is allosterically activated by free polyubiquitin chains assigned to their equilibrium binding to the ubiquitin-interacting motif (UIM) at helix 17, and the approximately 1.4-fold increase in Vmax and slightly increased affinity for Glc [30] may be explained by a destabilization of the interaction between this helix and helix Moreover, deletion of the C-terminal helix results in a catalytically completely inactive enzyme [30] Two of the substitutions studied (K169N and E256K) are naturally occurring mutations in the GCK gene associated with familial, mild fasting hyperglycemia (MODY2), and the information provided by the present study is expected to represent a valuable reference for further studies on specific mutations in the GCK gene in which the molecular mechanism of the hyperglycemia is not yet understood [27] Experimental procedures Materials The QuickChangeÒ XL Site-Directed Mutagenesis Kit was obtained from Stratagene (La Jolla, CA, USA) The Big DyeÒ terminator v1.1 cycle sequencing kit used to prepare DNA for automated sequencing was provided by Applied Biosystems (Foster City, CA, USA) The oligonucleotide primers used for site-directed mutagenesis and sequencing were obtained from Invitrogen (Carlsbad, CA, USA) The restriction protease factor Xa was obtained from Protein Engineering Technology ApS (Aarhus, Denmark) Glutathione Sepharos 4B and SephadexƠ G-25 were purchased from Amersham Biosciences (GE Healthcare Europe GMBH, Oslo, Norway) Glc was purchased from 2478 Calbiochem (San Diego, CA, USA) GST, glucose 6-phosphate dehydrogenase, b-nicotinamide adenine dinucleotide, guanidium hydrochloride, ATP, dithiothreitol and magnesium chloride were obtained from Sigma-Aldrich (St Louis, MO, USA) MH was obtained from Glycoteam GmbH (Hamburg, Germany) The Centricon centrifugal filter unit was obtained from Millipore (Bedford, MA, USA) All chemicals and buffers used for fluorescence measurements were of the highest grade available Site-directed mutagenesis The mutations W99F, W167F, W257F, T168G, K169N, N204A, D205A, N231A, E256A ⁄ K, Q287V and E290A were introduced into the wild-type hGK (isoform1) cDNA using the QuikChangeÒ XL Site-Directed Mutagenesis Kit and the specific oligonucleotide primers listed in supplementary Table S2 The pGEX-3X vector (kindly provided by F M Matschinsky, University of Pennsylvania, USA), containing the restriction protease factor Xa cleavage site, was used as the template host DNA sequencing was used to verify the introduction of the desired mutations Expression and purification of recombinant hGK The wild-type and mutant forms of hGK isoform were expressed as GST fusion proteins Expression in Escherichia coli (BL21) cells at 28 °C and purification of proteins by glutathione Sepharose 4B affinity chromatography were performed as previously reported ($ 10 mg of soluble protein per L of culture for the wild-type and 5–10 mg for the mutant forms) [30] Wild-type hGK and selected mutant forms were further purified by removing the GST fusion protein after cleavage for h at °C by factor Xa at a protease to substrate ratio of : 25 (by mass) Glutathione and salts were removed by size exclusion chromatography (Sephadex G-25), followed by glutathione Sepharose 4B affinity chromatography to retain free GST and any uncleaved GST-hGK All purification steps were performed at °C in the presence of mm dithiothreitol The proteins were concentrated, aliquoted and stored in liquid nitrogen The recombinant proteins were isolated to a purity of > 98% (SDS ⁄ PAGE) for both GST-hGK and hGK, with an expected molecular mass of 76 kDa and 50 kDa, respectively, as previously described for wild-type hGK [30] Protein concentrations were determined using A280 (1 mgỈmL)1Ỉcm)1) of 1.05 for the wild-type GST-hGK, determined according to the method of Gill and von Hippel [31] in 0.02 m phosphate buffer (pH 6.5) with or without m guanidium hydrochloride For the mutants W99F, W167F and W257F GST-hGK, A280 (1 mgỈmL)1) of 0.97 was used For the isolated wild-type protein (without fusion partner), A280 of 0.65 was used whereas, for the isolated Trp mutants, A280 of 0.54 was used FEBS Journal 275 (2008) 2467–2481 ª 2008 The Authors Journal compilation ª 2008 FEBS J Molnes et al Activation of glucokinase by D-glucose binding Enzymatic assays The catalytic activity of GK was measured spectrophotometrically (A340nm) at 37 °C by a glucose 6-phosphate dehydrogenase coupled assay in a reaction mixture (1 mL) containing 25 mm Hepes (pH 7.4), 25 mm KCl, 7.5 mm MgCl2, mm dithiothreitol, 0.1% (w ⁄ v) BSA, mm ATP, 1.0 mm NAD+, 0.35 U glucose 6-phosphate dehydrogenase and varying concentrations of Glc The reaction was initiated by 0.5 lg of hGK (pre-incubated with Glc and mm dithiothreitol for 10 min); for mutants with reduced activity, the amount of protein was correspondingly increased (up to 200-fold) Reaction rates were calculated from linear regression of the change in A340nm To determine the kinetic variables, 12–15 dilutions of Glc (0–200 mm) were used but, when analysing very low-affinity active site mutants, the concentration range was 0–1600 mm Glc Nonlinear regression analyses of the experimental data using the Hill equation were applied to calculate the [S]0.5 value for Glc and nH Intrinsic tryptophan fluorescence measurements Fluorescence measurements were performed on a PerkinElmer LS-50B instrument (1 cm path-length quartz cell with maximal stirring; Perkin-Elmer, Waltham, MA, USA) at 25 °C (constant temperature cell holder) in a buffer containing 20 mm Hepes, 100 mm NaCl and mm dithiothreitol (pH 7) The steady-state emission spectra were recorded from 305–500 nm with a fixed excitation wavelength of 295 nm, slit widths for excitation and emission of and nm, respectively, and by averaging four scans To denature hGK, the protein was incubated with m guanidium hydrochloride overnight To study the effect of substrate (Glc) and substrate analogue (MH), the change in fluorescence intensity (DFeq ⁄ F0 values at kmax) was measured as a function of the concentration of added ligand A concentration range of 0–600 mm Glc and 0–40 mm MH was used in the titrations The observed fluorescence was corrected for background emission (< 5%) and dilutions due to ligand addition, which in most cases did not exceed 10% Nonlinear regression analysis of the data (the response ⁄ binding isotherm) was performed using sigmaplot technical graphing Software (Systat Software Inc., San Jose, CA, USA) and the equation: DFeq =F0 ẳ DFeq =F0 ịmax ẵS=Kd ỵ ẵSị ð2Þ where F0 is the fluorescence baseline value, DFeq is the equilibrium fluorescence response, [S] is the ligand concentration and Kd is the equilibrium dissociation constant defined as the ligand concentration of half maximal increase in fluorescence intensity Temperature-induced changes in ITF were followed in the range 7–37 °C The sample temperature was controlled by a thermistor (ETI 2002; Electronic Temperature Instruments Ltd., Worthing, UK) with an accuracy of ± 0.2 °C, and the cell compartment was flushed with N2 The time course for the Glc-induced fluorescence enhancement (DFeq ⁄ F0) was followed with excitation and emission slit widths of and nm, respectively To determine the enthalpy and entropy change of the hGK-Glc interaction, the apparent equilibrium dissociation constant (Kd) was measured at six different temperatures, in the range 7– 32 °C at approximately °C intervals Van’t Hoff analysis was carried out assuming that DH and DS of the hGK-Glc interaction vary negligibly with temperature, using the equation: ln Kd ¼ DHvan0 t Hoff =RT À DS=R 3ị where R is the universal gas constant (8.31 Jặmol)1ặK)1) and T is the absolute temperature in kelvin CD spectroscopy Far-UV CD spectra (185–260 nm, light path mm) were recorded at 25 °C on a Jasco J-810 spectropolarimeter equipped with a Peltier element for temperature control The isolated wild-type and W167F proteins were diluted in a 20 mm sodium phosphate buffer (pH 7) to a final concentration of 15 lm Each spectrum obtained was an average of four scans at a scan rate of 50 nmỈmin)1 The resultant spectra were background-corrected and smoothed The secondary structure elements of the proteins were evaluated by the CD Neural Network algorithm [32] The global conformational stability, as measured by thermal denaturation (5–90 °C), was determined by measuring the change in ellipticity at 222 nm at a constant scanning rate of 40 °CỈh)1 The apparent transition temperature (Tm) was determined from the first derivative of the smoothed denaturation curve Structural analyses In the present study, the MolMovDB of The Yale Morph Server (http://molmovdb.mbb.yale.edu/molmovdb/) [33,34] was used to demonstrate the regions of variable secondary structure in the two determined crystal structures of hGK [12] Seventeen helices were identified in the ligand-free super-open structure (PDB identity: 1v4t) versus 19 helices in the Glc and allosteric activator compound A bound fully-closed structure (PDB identity: 1v4s); H17 (residues 447–460) fi H19 (residues 443–461) (http://molmovdb mbb.yale.edu/cgi-bin/morph.cgi?ID=496337–23316) The Molmovdb was also used to predict hinge regions (Hinge Master algorithm) and to calculate the changes in the backbone dihedral torsion angles (Du + Dw) for selected motifs ⁄ residues upon binding of Glc based on the coordinates of the unliganded and the liganded form Helix–helix interactions in the two conformational states were analysed as described previously [35] The static solvent accessibility of individual residues in the two conformational states was FEBS Journal 275 (2008) 2467–2481 ª 2008 The Authors Journal compilation ª 2008 FEBS 2479 Activation of glucokinase by D-glucose binding J Molnes et al calculated using an algorithm described previously [16] Global motion analysis of the two conformational states was performed with the GNM [20,21] sigmaplot graphical software was used to visualize the GNM output files for all residues Structural images were generated using pymol, version 0.99 (http://www.pymol.org) Acknowledgements This work was supported by Helse Vest, Haukeland University Hospital, the Norwegian Research Council, the Novo Nordisk Foundation, the Norwegian Diabetes Association, the University of Bergen, and the Meltzer Foundation We thank Anita-Merete Nordbø for expert technical assistance, Ali Sepulveda Munoz ˜ for French press preparation of recombinant enzyme and Ingvild Aukrust and Joao Barroso for introduc˜ tion to the methods of CD and ITF spectroscopy, respectively References Nishi S, Stoffel M, Xiang K, Shows TB, Bell GI & Takeda J (1992) Human pancreatic beta-cell glucokinase: cDNA sequence and localization of the polymorphic gene to chromosome 7, band p 13 Diabetologia 35, 743–747 Matschinsky FM (2002) Regulation of pancreatic betacell glucokinase: from basics to therapeutics Diabetes 51(Suppl 3), S394–S404 Tanizawa Y, Koranyi LI, Welling CM & Permutt MA (1991) Human liver glucokinase gene: cloning and sequence determination of two alternatively spliced cDNAs Proc Natl Acad Sci U S A 88, 7294– 7297 Hughes SD, Quaade C, Milburn JL, Cassidy L & Newgard CB (1991) Expression of normal and novel glucokinase mRNAs in anterior pituitary and islet cells J Biol Chem 266, 4521–4530 Gloyn AL (2003) Glucokinase (GCK) mutations in hyper- and hypoglycemia: maturity-onset diabetes of the young, permanent neonatal diabetes, and hyperinsulinemia of infancy Hum Mutat 22, 353– 362 Njølstad PR, Sagen JV, Bjørkhaug L, Odili S, Shehadeh N, Bakry D, Sarici SU, Alpay F, Molnes J, Molven A et al (2003) Permanent neonatal diabetes caused by glucokinase deficiency: inborn error of the glucose-insulin signaling pathway Diabetes 52, 2854–2860 Njølstad PR, Søvik O, Cuesta-Munoz A, Bjørkhaug L, Massa O, Barbetti F, Undlien DE, Shiota C, Magnuson MA, Molven A et al (2001) Neonatal diabetes mellitus due to complete glucokinase deficiency N Engl J Med 344, 1588–1592 2480 Cornish-Bowden A & Storer AC (1986) Mechanistic origin of the sigmoidal rate behaviour of rat liver hexokinase D (‘glucokinase’) Biochem J 240, 293–296 Storer AC & Cornish-Bowden A (1977) Kinetic evidence for a ‘mnemonical’ mechanism for rat liver glucokinase Biochem J 165, 61–69 10 Lin SX & Neet KE (1990) Demonstration of a slow conformational change in liver glucokinase by fluorescence spectroscopy J Biol Chem 265, 9670–9675 11 Heredia VV, Thomson J, Nettleton D & Sun S (2006) Glucose-induced conformational changes in glucokinase mediate allosteric regulation: transient kinetic analysis Biochemistry 45, 7553–7562 12 Kamata K, Mitsuya M, Nishimura T, Eiki J & Nagata Y (2004) Structural basis for allosteric regulation of the monomeric allosteric enzyme human glucokinase Structure 12, 429–438 13 Frieden C (1970) Kinetic aspects of regulation of metabolic processes The hysteretic enzyme concept J Biol Chem 245, 5788–5799 14 Davis EA, Cuesta-Munoz A, Raoul M, Buettger C, Sweet I, Moates M, Magnuson MA & Matschinsky FM (1999) Mutants of glucokinase cause hypoglycaemia- and hyperglycaemia syndromes and their analysis illuminates fundamental quantitative concepts of glucose homeostasis Diabetologia 42, 1175–1186 15 Liang Y, Kesavan P, Wang LQ, Niswender K, Tanizawa Y, Permutt MA, Magnuson MA & Matschinsky FM (1995) Variable effects of maturity-onset-diabetesof-youth (MODY)-associated glucokinase mutations on substrate interactions and stability of the enzyme Biochem J 309, 167–173 16 Parthiban V, Gromiha MM & Schomburg D (2006) CUPSAT: prediction of protein stability upon point mutations Nucleic Acids Res 34, W239–W242 17 Moukil MA & Van Schaftingen E (2001) Analysis of the cooperativity of human beta-cell glucokinase through the stimulatory effect of glucose on fructose phosphorylation J Biol Chem 276, 3872–3878 18 Moukil MA, Veiga-da-Cunha M & Van Schaftingen E (2000) Study of the regulatory properties of glucokinase by site-directed mutagenesis: conversion of glucokinase to an enzyme with high affinity for glucose Diabetes 49, 195–201 19 Brocklehurst KJ, Payne VA, Davies RA, Carroll D, Vertigan HL, Wightman HJ, Aiston S, Waddell ID, Leighton B, Coghlan MP et al (2004) Stimulation of hepatocyte glucose metabolism by novel small molecule glucokinase activators Diabetes 53, 535–541 20 Yang LW & Bahar I (2005) Coupling between catalytic site and collective dynamics: a requirement for mechanochemical activity of enzymes Structure 13, 893–904 21 Yang LW, Rader AJ, Liu X, Jursa CJ, Chen SC, Karimi HA & Bahar I (2006) oGNM: online computa- FEBS Journal 275 (2008) 2467–2481 ª 2008 The Authors Journal compilation ª 2008 FEBS J Molnes et al 22 23 24 25 26 27 28 29 30 tion of structural dynamics using the Gaussian Network Model Nucleic Acids Res 34, W24–W31 Heredia VV, Carlson TJ, Garcia E & Sun S (2006) Biochemical basis of glucokinase activation and the regulation by glucokinase regulatory protein in naturally occuring mutations J Biol Chem 281, 40201–40207 Zhang J, Li C, Chen K, Zhu W, Shen X & Jiang H (2006) Conformational transition pathway in the allosteric process of human glucokinase Proc Natl Acad Sci U S A 103, 13368–13373 Kim YB, Kalinowski SS & Marcinkeviciene J (2007) A pre-steady state analysis of ligand binding to human glucokinase: evidence for a preexisting equilibrium Biochemistry 46, 1423–1431 Aleshin AE, Zeng C, Bartunik HD, Fromm HJ & Honzatko RB (1998) Regulation of hexokinase I: crystal structure of recombinant human brain hexokinase complexed with glucose and phosphate J Mol Biol 282, 345–357 Gerstein M, Lesk AM & Chothia C (1994) Structural mechanisms for domain movements in proteins Biochemistry 33, 6739–6749 Matschinsky FM, Magnuson MA, Zelent D, Jetton TL, Doliba N, Han Y, Taub R & Grimsby J (2006) The network of glucokinase-expressing cells in glucose homeostasis and the potential of glucokinase activators for diabetes therapy Diabetes 55, 1–12 Pedelini L, Garcia-Gimeno MA, Marina A, GomezZumaquero JM, Rodriguez-Bada P, Lopez-Enriquez S, Soriguer FC, Cuesta-Munoz AL & Sanz P (2005) Structure-function analysis of the alpha5 and the alpha13 helices of human glucokinase: description of two novel activating mutations Protein Sci 14, 2080– 2086 Wabitsch M, Lahr G, Van de Bunt M, Marchant C, Lindner M, von Puttkamer J, Fenneberg A, Debatin KM, Klein R, Ellard S et al (2007) Heterogeneity in disease severity in a family with a novel G68V GCK activating mutation causing persistent hyperinsulinaemic hypoglycaemia of infancy Diabet Med 24, 1393–1399 Bjørkhaug L, Molnes J, Søvik O, Njølstad PR & Flatmark T (2007) Allosteric activation of human glucokinase by free polyubiquitin chains and its Activation of glucokinase by D-glucose binding 31 32 33 34 35 ubiquitin-dependent cotranslational proteasomal degradation J Biol Chem 282, 22757–22764 Gill SC & von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data Anal Biochem 182, 319–326 Bohm G, Muhr R & Jaenicke R (1992) Quantitative analysis of protein far UV circular dichroism spectra by neural networks Protein Eng 5, 191–195 Gerstein M & Krebs W (1998) A database of macromolecular motions Nucleic Acids Res 26, 4280–4290 Krebs WG & Gerstein M (2000) The morph server: a standardized system for analyzing and visualizing macromolecular motions in a database framework Nucleic Acids Res 28, 1665–1675 Burba AE, Lehnert U, Yu EZ & Gerstein M (2006) Helix Interaction Tool (HIT): a web-based tool for analysis of helix-helix interactions in proteins Bioinformatics 22, 2735–2738 Supplementary material The following supplementary material is available online: Fig S1 The time course for the d-glucose-induced fluorescence enhancement of wild-type hGK and its W fi F mutant forms Fig S2 Equilibrium binding of MH Fig S3 The ‘catalytic efficiency’ and the total Glcinduced fluorescence change of the active site mutants (fusion proteins) Table S1 Response of 200 mm glucose on intrinsic tryptophan fluorescence and catalytic activity of wildtype GST-hGK and W fi F mutant forms Table S2 Oligonucleotides used for PCR-based mutagenesis This material is available as part of the online article from http://www.blackwell-synergy.com Please note: Blackwell Publishing are not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corresponding author for the article FEBS Journal 275 (2008) 2467–2481 ª 2008 The Authors Journal compilation ª 2008 FEBS 2481 ... involved in the binding of Glc to the super-open state (Fig 1C) of this two domain [large (L) and small (S)] enzyme, and thus the site of initiation of the global conformational transition; and (c) to. .. T168 and K169) (Fig 1B) To identify the contact residues involved in the initial binding of Glc to the super-open state (Fig 1C) of this two domain hinge-bending enzyme, all the actual residues... studies are required to settle the issue of a possible MgATP2) induced conformational change in GK Residues involved in the binding of D-glucose to the super-open conformation The 3D structure has