RESUMEN
BACKGROUND: Amino acid substitutions can perturb protein activity in multiple ways. Understanding their mechanistic basis may pinpoint how residues contribute to protein function. Here, we characterize the mechanisms underlying variant effects in human glucokinase (GCK) variants, building on our previous comprehensive study on GCK variant activity. RESULTS: Using a yeast growth-based assay, we score the abundance of 95% of GCK missense and nonsense variants. When combining the abundance scores with our previously determined activity scores, we find that 43% of hypoactive variants also decrease cellular protein abundance. The low-abundance variants are enriched in the large domain, while residues in the small domain are tolerant to mutations with respect to abundance. Instead, many variants in the small domain perturb GCK conformational dynamics which are essential for appropriate activity. CONCLUSIONS: In this study, we identify residues important for GCK metabolic stability and conformational dynamics. These residues could be targeted to modulate GCK activity, and thereby affect glucose homeostasis.
Asunto(s)
Diabetes Mellitus Tipo 2 , Glucoquinasa , Humanos , Sustitución de Aminoácidos , Diabetes Mellitus Tipo 2/genética , Glucoquinasa/genética , Glucoquinasa/química , Glucoquinasa/metabolismo , MutaciónRESUMEN
The hole mutagenesis approach was used to interrogate the importance of F337 in Trypanosoma cruzi glucokinase (TcGlcK) in order to understand the complete set of binding interactions that are made by d-glucosamine analogue inhibitors containing aromatic tail groups that can extend to the outer part of the active site. An interesting inhibitor of this analogue class includes 2-N-carboxybenzyl-2-deoxy-d-glucosamine (CBZ-GlcN), which exhibits strong TcGlcK binding with a Ki of 710 nM. The residue F337 is found at the outer part of the active site that stems from the second protein subunit of the homodimeric assembly. In this study, F337 was changed to leucine and alanine so as to diminish phenylalanine's side chain size and attenuate intermolecular interactions in this region of the binding cavity. Results from enzyme - inhibitor assays revealed that the phenyl group of F337 made dominant hydrophobic interactions with the phenyl group of CBZ-GlcN as opposed to π - π stacking interactions. Moreover, enzymatic activity assays and X-ray crystallographic experiments indicated that each of these site-directed mutants primarily retained their activity and had high structural similarity of their protein fold. A computed structure model of T. cruzi hexokinase (TcHxK), which was produced by the artificial intelligence system AlphaFold, was compared to an X-ray crystal structure of TcGlcK. Our structural analysis revealed that TcHxK lacked an F337 counterpart residue and probably exists in the monomeric form. We proposed that the d-glucosamine analogue inhibitors that are structurally similar to CBZ-GlcN may not bind as strongly in TcHxK as they do in TcGlcK because of absent van der Waals contact from residue side chains.
Asunto(s)
Enfermedad de Chagas , Trypanosoma cruzi , Humanos , Glucoquinasa/química , Glucoquinasa/metabolismo , Dominio Catalítico , Fenilalanina , Inteligencia Artificial , Modelos Moleculares , Glucosamina , Sitios de Unión , Cristalografía por Rayos XRESUMEN
Although ADP-dependent sugar kinases were first described in archaea, at present, the presence of an ADP-dependent glucokinase (ADP-GK) in mammals is well documented. This enzyme is mainly expressed in hematopoietic lineages and tumor tissues, although its role has remained elusive. Here, we report a detailed kinetic characterization of the human ADP-dependent glucokinase (hADP-GK), addressing the influence of a putative signal peptide for endoplasmic reticulum (ER) destination by characterizing a truncated form. The truncated form revealed no significant impact on the kinetic parameters, showing only a slight increase in the Vmax value, higher metal promiscuity, and the same nucleotide specificity as the full-length enzyme. hADP-GK presents an ordered sequential kinetic mechanism in which MgADP is the first substrate to bind and AMP is the last product released, being the same mechanism described for archaeal ADP-dependent sugar kinases, in agreement with the protein topology. Substrate inhibition by glucose was observed due to sugar binding to nonproductive species. Although Mg2+ is an essential component for kinase activity, it also behaves as a partial mixed-type inhibitor for hADP-GK, mainly by decreasing the MgADP affinity. Regarding its distribution, phylogenetic analysis shows that ADP-GK's are present in a wide diversity of eukaryotic organisms although it is not ubiquitous. Eukaryotic ADP-GKs sequences cluster into two main groups, showing differences in the highly conserved sugar-binding motif reported for archaeal enzymes [NX(N)XD] where a cysteine residue is found instead of asparagine in a significant number of enzymes. Site directed mutagenesis of the cysteine residue by asparagine produces a 6-fold decrease in Vmax, suggesting a role for this residue in the catalytic process, probably by facilitating the proper orientation of the substrate to be phosphorylated.
Asunto(s)
Asparagina , Cisteína , Humanos , Adenosina Difosfato/metabolismo , Secuencia de Aminoácidos , Glucoquinasa/química , Glucosa/metabolismo , Cinética , Filogenia , AzúcaresRESUMEN
BACKGROUND: Glucokinase (GCK) regulates insulin secretion to maintain appropriate blood glucose levels. Sequence variants can alter GCK activity to cause hyperinsulinemic hypoglycemia or hyperglycemia associated with GCK-maturity-onset diabetes of the young (GCK-MODY), collectively affecting up to 10 million people worldwide. Patients with GCK-MODY are frequently misdiagnosed and treated unnecessarily. Genetic testing can prevent this but is hampered by the challenge of interpreting novel missense variants. RESULT: Here, we exploit a multiplexed yeast complementation assay to measure both hyper- and hypoactive GCK variation, capturing 97% of all possible missense and nonsense variants. Activity scores correlate with in vitro catalytic efficiency, fasting glucose levels in carriers of GCK variants and with evolutionary conservation. Hypoactive variants are concentrated at buried positions, near the active site, and at a region of known importance for GCK conformational dynamics. Some hyperactive variants shift the conformational equilibrium towards the active state through a relative destabilization of the inactive conformation. CONCLUSION: Our comprehensive assessment of GCK variant activity promises to facilitate variant interpretation and diagnosis, expand our mechanistic understanding of hyperactive variants, and inform development of therapeutics targeting GCK.
Asunto(s)
Diabetes Mellitus Tipo 2 , Glucoquinasa , Humanos , Glucoquinasa/genética , Glucoquinasa/química , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/diagnóstico , Mutación Missense , Pruebas Genéticas , MutaciónRESUMEN
Glucokinase activators are considered as new therapeutic arsenals that bind to the allosteric activator sites of glucokinase enzymes, thereby maximizing its catalytic rate and increasing its affinity to glucose. This study was designed to identify potent glucokinase activators from prenylated flavonoids isolated from medicinal plants using molecular docking, molecular dynamics simulation, density functional theory, and ADMET analysis. Virtual screening was carried out on glucokinase enzymes using 221 naturally occurring prenylated flavonoids, followed by molecular dynamics simulation (100 ns), density functional theory (B3LYP model), and ADMET (admeSar 2 online server) studies. The result obtained from the virtual screening with the glucokinase revealed arcommunol B (-10.1 kcal/mol), kuwanon S (-9.6 kcal/mol), manuifolin H (-9.5 kcal/mol), and kuwanon F (-9.4 kcal/mol) as the top-ranked molecules. Additionally, the molecular dynamics simulation and MM/GBSA calculations showed that the hit molecules were stable at the active site of the glucokinase enzyme. Furthermore, the DFT and ADMET studies revealed the hit molecules as potential glucokinase activators and drug-like candidates. Our findings suggested further evaluation of the top-ranked prenylated flavonoids for their in vitro and in vivo glucokinase activating potentials.
Asunto(s)
Activadores de Enzimas/farmacología , Flavonoides/farmacología , Glucoquinasa/metabolismo , Dominio Catalítico/efectos de los fármacos , Activadores de Enzimas/química , Flavonoides/química , Glucoquinasa/química , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica MolecularRESUMEN
Diabetes mellitus is a global threat affecting millions of people of different age groups. In recent years, the development of naturally derived anti-diabetic agents has gained popularity. Okra is a common vegetable containing important bioactive components such as abscisic acid (ABA). ABA, a phytohormone, has been shown to elicit potent anti-diabetic effects in mouse models. Keeping its anti-diabetic potential in mind, in silico study was performed to explore its role in inhibiting proteins relevant to diabetes mellitus- 11ß-hydroxysteroid dehydrogenase (11ß-HSD1), aldose reductase, glucokinase, glutamine-fructose-6-phosphate amidotransferase (GFAT), peroxisome proliferator-activated receptor-gamma (PPAR-gamma), and Sirtuin family of NAD(+)-dependent protein deacetylases 6 (SIRT6). A comparative study of the ABA-protein docked complex with already known inhibitors of these proteins relevant to diabetes was compared to explore the inhibitory potential. Calculation of molecular binding energy (ΔG), inhibition constant (pKi), and prediction of pharmacokinetics and pharmacodynamics properties were performed. The molecular docking investigation of ABA with 11-HSD1, GFAT, PPAR-gamma, and SIRT6 revealed considerably low binding energy (ΔG from -8.1 to -7.3 Kcal/mol) and predicted inhibition constant (pKi from 6.01 to 5.21 µM). The ADMET study revealed that ABA is a promising drug candidate without any hazardous effect following all current drug-likeness guidelines such as Lipinski, Ghose, Veber, Egan, and Muegge.
Asunto(s)
Abelmoschus/química , Ácido Abscísico/farmacología , Diabetes Mellitus/metabolismo , Hipoglucemiantes/farmacología , Proteínas/metabolismo , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/antagonistas & inhibidores , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/química , 11-beta-Hidroxiesteroide Deshidrogenasa de Tipo 1/metabolismo , Ácido Abscísico/química , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacocinética , Aldehído Reductasa/química , Aldehído Reductasa/metabolismo , Simulación por Computador , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Glucoquinasa/química , Glucoquinasa/metabolismo , Glutamina/metabolismo , Glutamina-Fructosa-6-Fosfato Transaminasa (Isomerizadora)/metabolismo , Glucógeno Sintasa Quinasa 3/química , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Hipoglucemiantes/química , Simulación del Acoplamiento Molecular , PPAR gamma/química , PPAR gamma/metabolismo , Proteínas/química , Sirtuinas/química , Sirtuinas/metabolismoRESUMEN
The investigation of allosteric effects in biomolecular structures is of great current interest in diverse areas, from fundamental biological enquiry to drug discovery. Here we present ProteinLens, a user-friendly and interactive web application for the investigation of allosteric signalling based on atomistic graph-theoretical methods. Starting from the PDB file of a biomolecule (or a biomolecular complex) ProteinLens obtains an atomistic, energy-weighted graph description of the structure of the biomolecule, and subsequently provides a systematic analysis of allosteric signalling and communication across the structure using two computationally efficient methods: Markov Transients and bond-to-bond propensities. ProteinLens scores and ranks every bond and residue according to the speed and magnitude of the propagation of fluctuations emanating from any site of choice (e.g. the active site). The results are presented through statistical quantile scores visualised with interactive plots and adjustable 3D structure viewers, which can also be downloaded. ProteinLens thus allows the investigation of signalling in biomolecular structures of interest to aid the detection of allosteric sites and pathways. ProteinLens is implemented in Python/SQL and freely available to use at: www.proteinlens.io.
Asunto(s)
Proteínas/química , Programas Informáticos , Regulación Alostérica , Sitio Alostérico , ADN/química , Glucoquinasa/química , Humanos , Internet , Conformación ProteicaRESUMEN
ADP-dependent kinases were first described in archaea, although their presence has also been reported in bacteria and eukaryotes (human and mouse). This enzyme family comprises three substrate specificities; specific phosphofructokinases (ADP-PFKs), specific glucokinases (ADP-GKs), and bifunctional enzymes (ADP-PFK/GK). Although many structures are available for members of this family, none exhibits fructose-6-phosphate (F6P) at the active site. Using an ancestral enzyme, we obtain the first structure of an ADP-dependent kinase (AncMsPFK) with F6P at its active site. Key residues for sugar binding and catalysis were identified by alanine scanning, D36 being a critical residue for F6P binding and catalysis. However, this residue hinders glucose binding because its mutation to alanine converts the AncMsPFK enzyme into a specific ADP-GK. Residue K179 is critical for F6P binding, while residues N181 and R212 are also important for this sugar binding, but to a lesser extent. This structure also provides evidence for the requirement of both substrates (sugar and nucleotide) to accomplish the conformational change leading to a closed conformation. This suggests that AncMsPFK mainly populates two states (open and closed) during the catalytic cycle, as reported for specific ADP-PFK. This situation differs from that described for specific ADP-GK enzymes, where each substrate independently causes a sequential domain closure, resulting in three conformational states (open, semiclosed, and closed).
Asunto(s)
Proteínas Arqueales/química , Fructosafosfatos/química , Glucoquinasa/química , Methanosarcinales/química , Fosfofructoquinasas/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Sitios de Unión , Biocatálisis , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Fructosafosfatos/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glucoquinasa/genética , Glucoquinasa/metabolismo , Cinética , Ligandos , Methanosarcinales/enzimología , Methanosarcinales/genética , Modelos Moleculares , Fosfofructoquinasas/genética , Fosfofructoquinasas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
The cooperativity of a monomeric enzyme arises from dynamic correlation instead of spatial correlation and is a consequence of nonequilibrium conformation fluctuations. We investigate the conformation-modulated kinetics of human glucokinase, a monomeric enzyme with important physiological functions, using a five-state kinetic model. We derive the non-Michealis-Menten (MM) correction term of the activity (i.e., turnover rate), predict its relationship to cooperativity, and reveal the violation of conformational detailed balance. Most importantly, we reproduce and explain the observed resonance effect in human glucokinase (i.e., maximal cooperativity when the conformational fluctuation rate is comparable to the catalytic rate). With the realistic parameters, our theoretical results are in quantitative agreement with the reported measurement by Miller and co-workers. The analysis can be extended to a general chemical network beyond the five-state model, suggesting the generality of kinetic cooperativity and resonance.
Asunto(s)
Glucoquinasa/metabolismo , Biocatálisis , Glucoquinasa/química , Glucosa/metabolismo , Humanos , Cinética , Conformación ProteicaRESUMEN
Glucokinase (GLK) and Hexokinase (HK) have been characterized as essential targets in Trypanosoma cruzi (Tc)-mediated infection. A recent study reported the propensity of the concomitant inhibition of TcGLK and TcHK by compounds GLK2-003 and GLK2-004, thereby presenting an efficient approach in Chagas disease treatment. We investigated this possibility using atomic and molecular scaling methods. Sequence alignment of TcGLK and TcHK revealed that both proteins shared approximately 33.3 % homology in their glucose/inhibitor binding sites. The total binding free energies of GLK2-003 and GLK2-004 were favorable in both proteins. PRO92 and THR185 were pivotal to the binding and stabilization of the ligands in TcGLK, likewise their conserved counterparts, PRO163 and THR237 in TcHK. Both compounds also induced a similar pattern of perturbations in both TcGLK and TcHK secondary structure. Findings from this study therefore provide insights into the underlying mechanisms of dual inhibition exhibited by the compounds. These results can pave way to discover and optimize novel dual Tc inhibitors with favorable pharmacokinetics properties eventuating in the mitigation of Chagas disease.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Glucoquinasa/antagonistas & inhibidores , Hexoquinasa/antagonistas & inhibidores , Tripanocidas/farmacología , Trypanosoma cruzi/enzimología , Enfermedad de Chagas/tratamiento farmacológico , Enfermedad de Chagas/parasitología , Inhibidores Enzimáticos/química , Glucoquinasa/química , Glucoquinasa/metabolismo , Hexoquinasa/química , Hexoquinasa/metabolismo , Humanos , Modelos Moleculares , Termodinámica , Tripanocidas/química , Trypanosoma cruzi/efectos de los fármacosRESUMEN
The genome sequence of Thermococcus kodakarensis contains an open reading frame, TK1110, annotated as ADP-dependent glucokinase. The encoding gene was expressed in Escherichia coli and the gene product, TK-GLK, was produced in soluble and active form. The recombinant enzyme was extremely thermostable. Thermostability was increased significantly in the presence of ammonium sulfate. ADP was the preferred co-factor for TK-GLK, which could be replaced with CDP but with a 60% activity. TK-GLK was a metal ion-dependent enzyme which exhibited glucokinase, glucosamine kinase and glucose 6-phosphatase activities. It catalyzed the phosphorylation of both glucose and glucosamine with nearly the same rate and affinity. The apparent Km values for glucose and glucosamine were 0.48 ± 0.03 and 0.47 ± 0.09 mM, respectively. The catalytic efficiency (kcat/Km) values against these two substrates were 6.2 × 105 ± 0.25 and 5.8 × 105 ± 0.75 M-1 s-1. The apparent Km value for dephosphorylation of glucose 6-phosphate was ~14-fold higher than that of glucose phosphorylation. Similarly, catalytic efficiency (kcat/Km) for phosphatase reaction was ~19-fold lower than that for the kinase reaction. To the best of our knowledge, this is the first report that describes the reversible nature of a euryarchaeal ADP-dependent glucokinase.
Asunto(s)
Adenosina Difosfato Glucosa/química , Adenosina Difosfato/química , Proteínas Arqueales/química , Glucoquinasa/química , Glucosamina/química , Glucosa/química , Thermococcus/enzimología , Adenosina Difosfato/metabolismo , Adenosina Difosfato Glucosa/metabolismo , Secuencia de Aminoácidos , Proteínas Arqueales/genética , Proteínas Arqueales/metabolismo , Sitios de Unión , Biocatálisis , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Glucoquinasa/genética , Glucoquinasa/metabolismo , Glucosamina/metabolismo , Glucosa/metabolismo , Cinética , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Thermococcus/química , TermodinámicaRESUMEN
The actin fold is found in cytoskeletal polymers, chaperones, and various metabolic enzymes. Many actin-fold proteins, such as the carbohydrate kinases, do not polymerize. We found that Glk1, a Saccharomyces cerevisiae glucokinase, forms two-stranded filaments with ultrastructure that is distinct from that of cytoskeletal polymers. In cells, Glk1 polymerized upon sugar addition and depolymerized upon sugar withdrawal. Polymerization inhibits enzymatic activity; the Glk1 monomer-polymer equilibrium sets a maximum rate of glucose phosphorylation regardless of Glk1 concentration. A mutation that eliminated Glk1 polymerization alleviated concentration-dependent enzyme inhibition. Yeast containing nonpolymerizing Glk1 were less fit when growing on sugars and more likely to die when refed glucose. Glk1 polymerization arose independently from other actin-related filaments and may allow yeast to rapidly modulate glucokinase activity as nutrient availability changes.
Asunto(s)
Actinas/química , Adenosina Trifosfatasas/química , Glucoquinasa/química , Hexoquinasa/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Adenosina Trifosfatasas/genética , Glucoquinasa/genética , Hexoquinasa/genética , Polimerizacion , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Glucose flux through glucokinase (GK) controls insulin release from the pancreas in response to high levels of glucose. Flux through GK is also responsible for reducing hepatic glucose output. Since many individuals with type 2 diabetes appear to have an inadequacy or defect in one or both of these processes, identifying compounds that can activate GK could provide a therapeutic benefit. Herein we report the further structure activity studies of a novel series of glucokinase activators (GKA). These studies led to the identification of pyridine 72 as a potent GKA that lowered post-prandial glucose in normal C57BL/6J mice, and after 14d dosing in ob/ob mice.
Asunto(s)
Activadores de Enzimas/química , Glucoquinasa/química , Hipoglucemiantes/química , Animales , Sitios de Unión , Glucemia/análisis , Cristalografía por Rayos X , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Activadores de Enzimas/metabolismo , Activadores de Enzimas/uso terapéutico , Glucoquinasa/metabolismo , Prueba de Tolerancia a la Glucosa , Hipoglucemiantes/metabolismo , Hipoglucemiantes/uso terapéutico , Cinética , Ratones , Ratones Endogámicos C57BL , Simulación de Dinámica Molecular , Relación Estructura-Actividad , Tiadiazoles/química , Tiadiazoles/metabolismoRESUMEN
Glucose phosphorylating enzymes are crucial in the regulation of basic cellular processes, including metabolism and gene expression. Glucokinases and hexokinases provide a pool of phosphorylated glucose in an adenosine diphosphate (ADP)- and ATP-dependent manner to shape the cell metabolism. The glucose processing enzymes from Kluyveromyces lactis are poorly characterized despite the emerging contribution of this yeast strain to industrial and laboratory scale biotechnology. The first reports on K. lactis glucokinase (KlGlk1) positioned the enzyme as an essential component required for glucose signaling. Nevertheless, no biochemical and structural information was available until now. Here, we present the first crystal structure of KlGlk1 together with biochemical characterization, including substrate specificity and enzyme kinetics. Additionally, comparative analysis of the presented structure and the prior structures of lactis hexokinase (KlHxk1) demonstrates the potential transitions between open and closed enzyme conformations upon ligand binding.
Asunto(s)
Glucoquinasa/química , Kluyveromyces/enzimología , Modelos Moleculares , Conformación Proteica , Glucoquinasa/genética , Glucoquinasa/metabolismo , Glucosa/metabolismo , Cinética , Kluyveromyces/genética , Kluyveromyces/metabolismo , Especificidad por SustratoRESUMEN
Proteomics-based mass spectrometry has gained increasing amounts of popularity in recent years. In particular, high resolution accurate mass measurements in mass spectrometry has gained notoriety for giving the capability of high throughput analysis with lower cost to the user. In particular, its uses in the identification of protein sequence through the utilization of bottom-up, middle-down, and top-down approaches has been widely discussed. In this chapter, we discuss the advantages of each technique as well as using the techniques in tandem to gain well-rounded structural data on our protein of interest, glucokinase. The study will focus on the use of Fourier-transform ion cyclotron resonance (FT-ICR) mass spectrometry, but give insights into the advantages that may come from the utilization of other high resolution techniques.
Asunto(s)
Glucoquinasa/química , Espectrometría de Masas , Análisis de Secuencia de Proteína , ProteómicaRESUMEN
Glucokinase (GK), a monomeric glucose-phosphorylating enzyme characterised by high structural flexibility, acts as a glucose sensor in pancreatic beta cells and liver. Pharmaceutical efforts to control the enzyme are hampered by an incomplete understanding of GK regulation. We investigated GK characteristics of wild-type and activating S64Y and G68V mutant proteins in the presence of various combinations of the synthetic activators RO-28-1675 and compound A, the endogenous activator fructose-2,6-bisphosphatase (FBPase-2), and the inhibitor mannoheptulose. S64Y impedes formation of a turn structure that is characteristic for the inactive enzyme conformation, and complex formation with compound A induces collision with the large domain. G68V evokes close contact of connecting region I and helix α13 with RO-28-1675 and compound A. Both mutants showed higher activity than the wild-type at low glucose and were susceptible to further activation by FBPase-2 and RO-28-1675, alone and additively. G68V was less active than S64Y, but was activatable by compound A. In contrast, compound A inhibited S64Y, and this effect was even more pronounced in combination with mannoheptulose. Mutant and wild-type GK showed comparable thermal stability and intracellular lifetimes. A GK-6-phosphofructo-2-kinase (PFK-2)/FBPase-2 complex predicted by in silico protein-protein docking demonstrated possible binding of the FBPase-2 domain near the active site of GK. In summary, activating mutations within the allosteric site of GK do not preclude binding of chemical activators (GKAs), but can alter their action into inhibition. Our postulated GK-PFK-2/FBPase-2 complex represents the endogenous principle of activation by substrate channelling which permits binding of other small molecules and proteins.
Asunto(s)
Glucoquinasa/metabolismo , Células Secretoras de Insulina/enzimología , Manoheptulosa/metabolismo , Proteínas Mutantes/metabolismo , Fosfofructoquinasa-2/metabolismo , Tiazoles/metabolismo , Sitio Alostérico , Animales , Dominio Catalítico , Línea Celular Tumoral , Glucoquinasa/química , Glucoquinasa/genética , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Manoheptulosa/química , Ratones , Fosfofructoquinasa-2/química , Unión Proteica , Conformación Proteica en Hélice alfa , Tiazoles/química , TransfecciónRESUMEN
Glucose phosphorylation by glucokinase exhibits a sigmoidal dependency on substrate concentration regardless of its simple structure. Dimorph mechanism suggested the existence of two enzymatic states with different catalytic properties, which has been shown to be plausible by structural analysis. However, the dimorph mechanism gives rise to a complicated or non-explicit non-closed mathematical form. It is neither feasible to apply the dimorph mechanism in effector characterizations. To improve the area of glucokinase study with stronger theoretical support and less complication in computation, we proposed the investigation of the enzyme from a pseudo-dimeric angle. The proposed mechanism started from the idealization of two monomeric glucokinase as a dimeric complex, which significantly simplified the glucose phosphorylation kinetics, while the differences in enzyme reconfiguration caused by variable substrates and effectors have been successfully characterized. The study presented a simpler and more reliable way in studying the properties of glucokinase and its effectors, providing guidelines of effector developments for hyperglycemia and hypoglycemia treatment.
Asunto(s)
Glucoquinasa/química , Glucosa/química , Modelos Químicos , Multimerización de Proteína , Catálisis , Humanos , FosforilaciónRESUMEN
Infection with the free-living amoeba Naegleria fowleri leads to life-threatening primary amoebic meningoencephalitis. Efficacious treatment options for these infections are limited, and the mortality rate is very high (â¼98%). Parasite metabolism may provide suitable targets for therapeutic design. Like most other organisms, glucose metabolism is critical for parasite viability, being required for growth in culture. The first enzyme required for glucose metabolism is typically a hexokinase (HK), which transfers a phosphate from ATP to glucose. The products of this enzyme are required for both glycolysis and the pentose phosphate pathway. However, the N. fowleri genome lacks an obvious HK homolog and instead harbors a glucokinase (Glck). The N. fowleri Glck (NfGlck) shares limited (25%) amino acid identity with the mammalian host enzyme (Homo sapiens Glck), suggesting that parasite-specific inhibitors with anti-amoeba activity can be generated. Following heterologous expression, NfGlck was found to have a limited hexose substrate range, with the greatest activity observed with glucose. The enzyme had apparent Km values of 42.5 ± 7.3 µM and 141.6 ± 9.9 µM for glucose and ATP, respectively. The NfGlck structure was determined and refined to 2.2-Å resolution, revealing that the enzyme shares greatest structural similarity with the Trypanosoma cruzi Glck. These similarities include binding modes and binding environments for substrates. To identify inhibitors of NfGlck, we screened a small collection of inhibitors of glucose-phosphorylating enzymes and identified several small molecules with 50% inhibitory concentration values of <1 µM that may prove useful as hit chemotypes for further leads and therapeutic development against N. fowleri.
Asunto(s)
Glucoquinasa/química , Glucoquinasa/metabolismo , Naegleria fowleri/enzimología , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Glucosa/metabolismo , Humanos , Trypanosoma cruzi/enzimologíaRESUMEN
Glucose metabolism in humans is tightly controlled by the activity of glucokinase (GCK). GCK is predominantly produced in the pancreas, where it catalyzes the rate-limiting step of insulin secretion, and in the liver, where it participates in glycogen synthesis. A multitude of disease-causing mutations within the gck gene have been identified. Activating mutations manifest themselves in the clinic as congenital hyperinsulinism, while loss-of-function mutations produce several diabetic conditions. Indeed, pharmaceutical companies have shown great interest in developing GCK-associated treatments for diabetic patients. Due to its essential role in maintaining whole-body glucose homeostasis, GCK activity is extensively regulated at multiple levels. GCK possesses a unique ability to self-regulate its own activity via slow conformational dynamics, which allows for a cooperative response to glucose. GCK is also subject to a number of protein-protein interactions and post-translational modification events that produce a broad range of physiological consequences. While significant advances in our understanding of these individual regulatory mechanisms have been recently achieved, how these strategies are integrated and coordinated within the cell is less clear. This review serves to synthesize the relevant findings and offer insights into the connections between molecular and cellular control of GCK.
Asunto(s)
Glucoquinasa/metabolismo , Animales , Proteínas Portadoras/fisiología , Activación Enzimática , Glucoquinasa/antagonistas & inhibidores , Glucoquinasa/química , Glucosa/análisis , Humanos , Fosfofructoquinasa-2/metabolismo , Unión Proteica , Conformación Proteica , Procesamiento Proteico-Postraduccional , Proteína SUMO-1/metabolismoRESUMEN
Glucokinase from pathogenic protozoa of the genus Leishmania is a potential drug target for the chemotherapeutic treatment against leishmaniasis because this enzyme is located at a nodal point between two critically important metabolic pathways, glycolysis and the pentose phosphate pathway (PPP). L. braziliensis glucokinase (LbGlcK) was evaluated for its structural characterization and enzymatic performance. The enzyme catalyzes the phosphorylation of d-glucose with co-substrate ATP to yield the products G6P and ADP. LbGlcK had KM values determined as 6.61 ± 2.63 mM and 0.338 ± 0.080 mM for d-glucose and ATP, respectively. The 1.85 Å resolution X-ray crystal structure of the apo form of LbGlcK was determined and a homodimer was revealed where each subunit (both in open conformations) included the typical small and large domains. Structural comparisons were assessed in relationship to Homo sapiens hexokinase IV and Trypanosoma cruzi glucokinase. Comparisons revealed that all residues important for making hydrogen bonding interactions with d-glucose in the active site and catalysis were strictly conserved. LbGlcK was screened against four glucosamine analogue inhibitors and the stronger inhibitor of the series, HPOP-GlcN, had a Ki value of 56.9 ± 16.6 µM that exhibited competitive inhibition. For the purpose of future structure-based drug design experimentation, L. braziliensis glucokinase was observed to be very similar to T. cruzi glucokinase even though there was a 44% protein sequence identity between the two enzymes.