RESUMEN
Recently we purified and identified a previously uncharacterized transcription factor from rat liver binding to the carbohydrate responsive element of the L-type pyruvate kinase (L-PK) gene. This factor was named carbohydrate responsive element binding protein (ChREBP). ChREBP, essential for L-PK gene transcription, is activated by high glucose and inhibited by cAMP. Here, we demonstrated that (i) nuclear localization signal and basic helix-loop-helix/leucine-zipper domains of ChREBP were essential for the transcription, and (ii) these domains were the targets of regulation by cAMP and glucose. Among three cAMP-dependent protein kinase phosphorylation sites, Ser(196) and Thr(666) were the target sites. Phosphorylation of the former resulted in inactivation of nuclear import, and that of the latter resulted in loss of the DNA-binding activity and L-PK transcription. On the other hand, glucose activated the nuclear import by dephosphorylation of Ser(196) in the cytoplasm and also stimulated the DNA-binding activity by dephosphorylation of Thr(666) in the nucleus. These results thus reveal mechanisms for regulation of ChREBP and the L-PK transcription by excess carbohydrate and cAMP.
Asunto(s)
AMP Cíclico/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación Enzimológica de la Expresión Génica , Glucosa/metabolismo , Secuencias Hélice-Asa-Hélice , Leucina Zippers , Piruvato Quinasa/genética , Sulfonamidas , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Sitios de Unión , Bucladesina/farmacología , Cantaridina/farmacología , Metabolismo de los Hidratos de Carbono , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , ADN/metabolismo , Proteínas de Unión al ADN/genética , Inhibidores Enzimáticos/farmacología , Isoquinolinas/farmacología , Masculino , Ratones , Datos de Secuencia Molecular , Mutagénesis , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Fosfoproteínas Fosfatasas/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas , Ratas , Ratas Sprague-Dawley , Elementos de Respuesta , Serina/genética , Serina/metabolismo , Fracciones Subcelulares , Treonina/genética , Treonina/metabolismo , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Carbohydrates mediate their conversion to triglycerides in the liver by promoting both rapid posttranslational activation of rate-limiting glycolytic and lipogenic enzymes and transcriptional induction of the genes encoding many of these same enzymes. The mechanism by which elevated carbohydrate levels affect transcription of these genes remains unknown. Here we report the purification and identification of a transcription factor that recognizes the carbohydrate response element (ChRE) within the promoter of the L-type pyruvate kinase (LPK) gene. The DNA-binding activity of this ChRE-binding protein (ChREBP) in rat livers is specifically induced by a high carbohydrate diet. ChREBP's DNA-binding specificity in vitro precisely correlates with promoter activity in vivo. Furthermore, forced ChREBP overexpression in primary hepatocytes activates transcription from the L-type Pyruvate kinase promoter in response to high glucose levels. The DNA-binding activity of ChREBP can be modulated in vitro by means of changes in its phosphorylation state, suggesting a possible mode of glucose-responsive regulation. ChREBP is likely critical for the optimal long-term storage of excess carbohydrates as fats, and may contribute to the imbalance between nutrient utilization and storage characteristic of obesity.
Asunto(s)
Metabolismo de los Hidratos de Carbono , Proteínas de Unión al ADN/fisiología , Glucosa/metabolismo , Hígado/metabolismo , Factores de Transcripción/fisiología , Animales , Secuencia de Bases , Células Cultivadas , Cartilla de ADN , Proteínas de Unión al ADN/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Secuencias Hélice-Asa-Hélice , Leucina Zippers , Masculino , Fosforilación , Regiones Promotoras Genéticas , Unión Proteica , Piruvato Quinasa/genética , Ratas , Ratas Sprague-Dawley , Factores de Transcripción/aislamiento & purificación , Factores de Transcripción/metabolismoRESUMEN
Macrophages can adapt to the absence of oxygen by switching to anaerobic glycolysis. In this study, we investigated (a) the roles of fructose 2,6-bisphosphate (Fru-2,6-P2) and ribose 1,5-bisphosphate (Rib-1,5-P2), potent activators of phosphofructokinase, (b) the enzymes responsible for the synthesis of Rib-1,5-P2, and (c) the mechanisms of regulation of these enzymes in H36.12j macrophages during the initial phase of hypoxia. Within 1 min after initiating hypoxia, glycolysis was activated through activation of phosphofructokinase. Over the same period, Fru-2,6-P2 decreased 50% and recovered completely upon reoxygenation. Similar changes in cAMP levels were observed. In contrast, the Rib-1,5-P2 concentration rapidly increased to a maximum level of 8.0 +/- 0.9 nmol/g cell 30 s after hypoxia. Thus, Rib-1,5-P2 was the major factor increasing the rate of glycolysis during the initial phase of hypoxia. Moreover, we found that Rib-1,5-P2 was synthesized by two steps: the ribose-phosphate pyrophosphokinase (5-phosphoribosyl-1-pyrophosphate synthetase; PRPP synthetase) reaction (EC ) catalyzing the reaction, Rib-5-P + ATP --> PRPP + AMP and a new enzyme, "PRPP pyrophosphatase" catalyzing the reaction, PRPP --> Rib-1,5-P2 + P(i). Both PRPP synthetase and PRPP pyrophosphatase were significantly activated 30 s after hypoxia. Pretreatment with 1-octadecyl-2-methyl-rac-glycero-3-phosphocholine and calphostin C prevented the activation of ribose PRPP synthetase and PRPP pyrophosphatase as well as increase in Rib-1,5-P2 and activation of phosphofructokinase 30 s after hypoxia. These data suggest that the activation of the above enzymes was mediated by protein kinase C acting via activation of phosphatidylinositol specific phospholipase C in the macrophages during hypoxia.
Asunto(s)
Fructosadifosfatos/metabolismo , Hipoxia , Macrófagos/metabolismo , Pentosafosfatos/fisiología , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Línea Celular , AMP Cíclico/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Calor , Cinética , Ratones , Modelos Biológicos , Naftalenos/farmacología , Oxígeno/metabolismo , Éteres Fosfolípidos/farmacología , Proteína Quinasa C/metabolismo , Ribosa-Fosfato Pirofosfoquinasa/metabolismo , Temperatura , Factores de TiempoRESUMEN
A bifunctional enzyme, fructose-6-phosphate, 2-kinase:fructose-2, 6-bisphosphatase, catalyzes synthesis and hydrolysis of fructose 2, 6-bisphosphate. The phosphatase reaction occurs in two steps: the formation of a phosphoenzyme intermediate and release of beta-D-fructose 6-phosphate, followed by hydrolysis of the phosphoenzyme. The objective of this study was to determine whether E325 in the Fru 2,6-Pase active site is an acid-base catalyst. The pH-rate profile for k(cat) for the wild-type enzyme exhibits pK values of 5.6 and 9.1. The pH dependence of k(cat) for the E325A mutant enzyme gives an increase in the acidic pK from 5.6 to 6.1. Formate, acetate, propionate, and azide accelerate the rate of hydrolysis of the E325A mutant enzyme, but not of the wild-type enzyme. Azide and formate, the smallest of the weak acids tested, are the most potent activators. The k(cat) vs pH profile of the E325A mutant enzyme in the presence of formate is similar to that of the wild-type enzyme. Taken together, these data are consistent with E325 serving an acid-base role in the phosphatase reaction. The exogenous low MW weak acids act as a replacement general base in the hydrolysis of the phosphoenzyme intermediate, rescuing some of the activity lost upon eliminating the glutamate side chain.
Asunto(s)
Ácido Glutámico/química , Monoéster Fosfórico Hidrolasas/química , Alanina/genética , Animales , Ácidos Carboxílicos/química , Catálisis , Activación Enzimática/genética , Formiatos/química , Ácido Glutámico/genética , Concentración de Iones de Hidrógeno , Cinética , Masculino , Peso Molecular , Fosfofructoquinasa-2 , Monoéster Fosfórico Hidrolasas/genética , Ratas , Azida Sódica/química , TestículoRESUMEN
Amino and carboxyl termini of the bifunctional enzyme Fru 6-P, 2-kinase:Fru 2,6-bisphosphatase regulate the relative activities of the kinase/phosphatase. The N-terminus of the rat liver bifunctional enzyme is highly basic, containing a protein kinase A phosphorylation site that regulates these enzyme activities in a reciprocal manner. To determine the role of charged residues in the N-terminal peptide, mutant enzymes were constructed in which these residues were altered to residues carrying opposite charges, and the effect on the catalytic properties, thermal lability, and susceptibility to trypsin digestion and phosphorylation by protein kinase A was determined. Most of these mutations decreased k(cat)/K(ATP) and/or k(cat)/K(Fru) (6-P) of the kinase and increased k(cat)/K(Fru 2,6-P2) of the phosphatase. These mutant enzymes were more susceptible to trypsin digestion, phosphorylation by protein kinase A, and thermal inactivation. In general, the effect was greater with amino acid residues located more distant from the N-terminus. The resulting changes were not as large as observed with the phosphorylated enzyme. Mutation of Ser22 to Pro produced large changes in the kinetic properties comparable to those of phosphorylation, suggesting that the flexible region of the N-terminus containing five serines (Ser20 to S24) is essential for the enzyme activities. These results indicated that the charged residues as well as Ser20-Ser24 in the N-terminus of the liver Fru 6-P,2-kinase:Fru 2,6-Pase are essential in the allosteric regulation and probably involved in interactions with the catalytic domains that induce a conformation that has high Fru 6-P,2-kinase and low Fru 2,6-Pase activities. Any disruption of this N-terminal interaction results in inhibition of the kinase and activation of the phosphatase.
Asunto(s)
Hígado/enzimología , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/metabolismo , Sustitución de Aminoácidos , Animales , Clonación Molecular , Cartilla de ADN , Escherichia coli , Cinética , Mutagénesis Sitio-Dirigida , Fosfofructoquinasa-2 , Monoéster Fosfórico Hidrolasas/genética , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase plays an essential role in the regulation of glucose metabolism by both producing and degrading Fru-2,6-P(2) via its distinct catalytic activities. The 6-PF-2-K and Fru-2,6-P(2)ase active sites are located in separate domains of the enzyme. The kinase domain is structurally related to the superfamily of mononucleotide binding proteins that includes adenylate kinase and the G-proteins. We have determined three new structures of the enzymatic monomer, each with a different ligand in the ATP binding site of the 6-PF-2-K domain (AMP-PNP, PO(4), and water). A comparison of these three new structures with the ATPgammaS-bound 6-PF-2-K domain reveals a rearrangement of a helix that is dependent on the ligand bound in the ATP binding site of the enzyme. This helix motion dramatically alters the position of a catalytic residue (Lys172). This catalytic cation is analogous to the Arg residue donated by the rasGAP protein, and the Arg residue at the core of the GTP or GDP sensing switch motion seen in the heterotrimeric G-proteins. In addition, a succinate molecule is observed in the Fru-6-P binding site. Kinetic analysis of succinate inhibition of the 6-PF-2-K reaction is consistent with the structural findings, and suggests a mechanism for feedback inhibition of glycolysis by citric acid cycle intermediates. Alterations in the 6-PF-2-K kinetics of several proteins mutated near both the switch and the succinate binding site suggest a mode of communication between the ATP- and F6P binding sites. Together with these kinetic data, these new structures provide insights into the mechanism of the 6-PF-2-K activity of this important bifunctional enzyme.
Asunto(s)
Dominio Catalítico , Fructosa-Bifosfatasa/química , Fructosa-Bifosfatasa/metabolismo , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Fosfofructoquinasa-1/química , Fosfofructoquinasa-1/metabolismo , Testículo/enzimología , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión , Cristalización , Cristalografía por Rayos X , Dimerización , Fructosafosfatos/metabolismo , Cinética , Masculino , Modelos Moleculares , Fosfofructoquinasa-2 , Fosfotransferasas/química , Fosfotransferasas/metabolismo , Estructura Secundaria de Proteína , Ratas , Succinatos/metabolismoRESUMEN
A bifunctional enzyme, fructose-6-phosphate,2-kinase/fructose 2, 6-bisphosphatase (Fru-6-P,2-kinase/Fru-2,6-Pase), catalyzes synthesis and degradation of fructose 2,6-bisphosphate (Fru-2,6-P2). Previously, the rat liver Fru-2,6-Pase reaction (Fru-2,6-P2 --> Fru-6-P + Pi) has been shown to proceed via a phosphoenzyme intermediate with His258 phosphorylated, and mutation of the histidine to alanine resulted in complete loss of activity (Tauler, A., Lin, K., and Pilkis, S. J. (1990) J. Biol. Chem. 265, 15617-15622). In the present study, it is shown that mutation of the corresponding histidine (His256) of the rat testis enzyme decreases activity by less than a factor of 10 with a kcat of 17% compared with the wild type enzyme. Mutation of His390 (in close proximity to His256) to Ala results in a kcat of 12.5% compared with the wild type enzyme. Attempts to detect a phosphohistidine intermediate with the H256A mutant enzyme were unsuccessful, but the phosphoenzyme is detected in the wild type, H390A, R255A, R305S, and E325A mutant enzymes. Data demonstrate that the mutation of His256 induces a change in the phosphatase hydrolytic reaction mechanism. Elimination of the nucleophilic catalyst, H256A, results in a change in mechanism. In the H256A mutant enzyme, His390 likely acts as a general base to activate water for direct hydrolysis of the 2-phosphate of Fru-2,6-P2. Mutation of Arg255 and Arg305 suggests that the arginines probably have a role in neutralizing excess charge on the 2-phosphate and polarizing the phosphoryl for subsequent transfer to either His256 or water. The role of Glu325 is less certain, but it may serve as a general acid, protonating the leaving 2-hydroxyl of Fru-2,6-P2.
Asunto(s)
Histidina/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Animales , Sitios de Unión , Dominio Catalítico , Histidina/genética , Cinética , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Mutagénesis Sitio-Dirigida , Fosfofructoquinasa-2 , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/genética , Fosforilación , Conformación Proteica , RatasRESUMEN
Fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase (Fru-6-P, 2-kinase/Fru-2,6-Pase) is a bifunctional enzyme, catalyzing the interconversion of beta-D-fructose- 6-phosphate (Fru-6-P) and fructose-2,6-bisphosphate (Fru-2,6-P2) at distinct active sites. A mutant rat testis isozyme with an alanine replacement for the catalytic histidine (H256A) in the Fru-2,6-Pase domain retains 17% of the wild type activity (Mizuguchi, H., Cook, P. F., Tai, C-H., Hasemann, C. A., and Uyeda, K. (1998) J. Biol. Chem. 274, 2166-2175). We have solved the crystal structure of H256A to a resolution of 2. 4 A by molecular replacement. Clear electron density for Fru-6-P is found at the Fru-2,6-Pase active site, revealing the important interactions in substrate/product binding. A superposition of the H256A structure with the RT2K-Wo structure reveals no significant reorganization of the active site resulting from the binding of Fru-6-P or the H256A mutation. Using this superposition, we have built a view of the Fru-2,6-P2-bound enzyme and identify the residues responsible for catalysis. This analysis yields distinct catalytic mechanisms for the wild type and mutant proteins. The wild type mechanism would lead to an inefficient transfer of a proton to the leaving group Fru-6-P, which is consistent with a view of this event being rate-limiting, explaining the extremely slow turnover (0. 032 s-1) of the Fru-2,6-Pase in all Fru-6-P,2-kinase/Fru-2,6-Pase isozymes.
Asunto(s)
Monoéster Fosfórico Hidrolasas/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Testículo/enzimología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Fructosadifosfatos/metabolismo , Masculino , Modelos Moleculares , Fosfofructoquinasa-2 , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Unión Proteica , Conformación Proteica , RatasRESUMEN
Transcription of the liver type pyruvate kinase and lipogenesis enzyme genes is induced by high carbohydrate in liver. We have found a novel protein factor in rat liver nuclei that binds to the glucose response element (CACGTG motifs) of the pyruvate kinase gene (Liu, Z. , Thompson, K. S., and Towle, H. C. (1993) J. Biol. Chem. 268, 12787-12795) and the "insulin response element" of fatty acid synthase gene. The amounts of this DNA-binding protein, termed "glucose response element binding protein" (GRBP) in the nuclear extract, were increased in liver by a high carbohydrate diet and decreased by starvation, high fat, and high protein diet. GRBP also occurs in cytosols of liver and is dependent on carbohydrate. Both the nuclear and the cytosolic GRBP showed similar properties, except the former was more resistant to thermal inactivation than the latter. Kinetics of glucose activation of the cytosolic GRBP in a primary culture of hepatocytes indicated that a half-maximum activation was achieved after 6 h, and glucose concentration required for the maximum activation of the GRBP was approximately 12 mM. Dibutyryl-cAMP, okadaic acid, and forskolin inhibited glucose activation of both GRBP and liver pyruvate kinase transcription. These results suggested that GRBP may be a factor that recognizes the glucose response motif site and may be involved in mediating carbohydrate response of the pyruvate kinase gene.
Asunto(s)
Ácido Graso Sintasas/metabolismo , Glucosa/metabolismo , Hígado/enzimología , Piruvato Quinasa/genética , Animales , Secuencia de Bases , Células Cultivadas , Colforsina/farmacología , AMP Cíclico/farmacología , Cartilla de ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Carbohidratos de la Dieta/administración & dosificación , Grasas de la Dieta/administración & dosificación , Regulación de la Expresión Génica/efectos de los fármacos , Glucosa/farmacología , Hígado/citología , Masculino , Datos de Secuencia Molecular , Ácido Ocadaico/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Site-directed mutagenesis was utilized to construct mutants, containing one or two tryptophan residues, of the bifunctional enzyme fructose 6-phosphate,2-kinase-fructose 2,6-bisphosphatase. Two of the single-tryptophan mutants (W15 and W64) had the tryptophan residue located in the kinase domain, which is in the N-terminal half, and two (W299 and W320) had the tryptophan residue located in the phosphatase domain, which is in the C-terminal half. The double-tryptophan mutants were W15/W64, W15/W299, W64/W299, and W299/W320. Dynamic polarization data indicated that these tryptophan residues had varying degrees of local mobility. Steady-state polarization data revealed energy transfer between the tryptophan residues in the double mutant W299/W320 but not in the W15/W64, W15/W299, or W64/W299 mutants, indicating the proximity of the W299 and W320 residues. The binding of fructose-6-phosphate resulted in a significant increase in the anisotropy of the W15 mutants, but did not affect the anisotropies of any of the other single-tryptophan mutants. Binding of fructose-2,6-bisphosphate also significantly increased the anisotropy of W15. In the case of fructose-6-phosphate binding, the increased anisotropy was shown to be due to a restriction of the tryptophan residue's local mobility in the presence of bound ligand, which suggests that the N-terminus is located near the kinase active site. These increases in anisotropies were used to estimate the dissociation constants of fructose-6-phosphate and fructose-2,6-bisphosphate, which were 29 +/- 3 and 2.1 +/- 0.3 microM, respectively. These observations are considered in light of the recently published crystal structure for this bifunctional enzyme.
Asunto(s)
Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Mutagénesis Sitio-Dirigida , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/genética , Fosfotransferasas/química , Fosfotransferasas/genética , Conformación Proteica , Testículo/enzimología , Sustitución de Aminoácidos/genética , Animales , Polarización de Fluorescencia , Fructosadifosfatos/metabolismo , Fructosafosfatos/metabolismo , Ligandos , Masculino , Fenilalanina/genética , Fosfofructoquinasa-2 , Unión Proteica , Ratas , Triptófano/genéticaRESUMEN
Fru 6-P,2-kinase:Fru 2,6-Pase is a bifunctional enzyme, consisting of highly conserved catalytic domains and variable regulatory domains. The regulatory domains reside in either the N- or the C-terminus, depending upon the isozyme. The rat testis enzyme (RT2K) lacks the regulatory domain, but the rat liver and the bovine heart enzymes contain phosphorylation site(s) in the N- and the C-termini, respectively. In order to determine whether the regulatory domains can be swapped, we have constructed mutant enzymes in which the N- or the C-terminal tail of the testis enzyme was replaced with that of either the liver or the heart enzyme. The substitution with the N-terminus of the liver enzyme (RLN-RT2K) resulted in a small change in the kinetic properties of Fru 6-P,2-kinase, but that with the heart enzyme increased the KFru 6-P 18-fold without affecting the Vmax. The substitution with the C-terminus of the heart enzyme had little effect. The phosphorylation of RLN-RT2K increased KFru 6-P fivefold as in the liver enzyme but did not affect the Fru 2,6-Pase, unlike the liver enzyme. All these mutant enzymes were more thermally labile than the wild type testis enzyme. RLN-RT2K was more sensitive to the denaturant. These results suggest that the N-terminus of the liver enzyme could interact with the kinase domain of the testis enzyme, regulating the kinase activity but was unable to affect the phosphatase domain. These differences could be explained by the large differences in net charges of the terminal tails.
Asunto(s)
Complejos Multienzimáticos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfotransferasas/metabolismo , Testículo/enzimología , Regulación Alostérica , Secuencia de Aminoácidos , Animales , Bovinos , Gluconatos/farmacología , Calor , Cinética , Hígado/enzimología , Masculino , Datos de Secuencia Molecular , Complejos Multienzimáticos/efectos de los fármacos , Complejos Multienzimáticos/genética , Miocardio/enzimología , Especificidad de Órganos , Fosfoenolpiruvato/farmacología , Fosfofructoquinasa-2 , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolasas/efectos de los fármacos , Monoéster Fosfórico Hidrolasas/genética , Fosfotransferasas/efectos de los fármacos , Fosfotransferasas/genética , Desnaturalización Proteica , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad , Urea/farmacologíaRESUMEN
A bifunctional enzyme, fructose-6-phosphate 2-kinase-fructose 2, 6-bisphosphatase, catalyzes synthesis and degradation of fructose 2, 6-bisphosphate. Mutants of basic residues, including Lys51, Arg78, Arg79, Arg136, Lys172, and Arg193, immediately around the active site of rat testis fructose 6-P,2-kinase were constructed, and their steady state kinetics, ATP binding, and the effect of pH on the kinetics were characterized. All mutants showed a several-fold increase in KMgATP, much larger increases in KFru 6-P, and decreased V compared to those of the wild type enzyme (WT). Replacement of Lys172 and Arg193 with Ala and Leu, respectively, also produced mutants with large KFru 6-P values. Substitution of Lys51, which is located in a Walker-A motif (GXXGXGKT, amino acids 45-52), with Ala or His resulted in enzymes with increased KMgATP values and unable to bind Fru 6-P. The dissociation constants for 2'(3')-O-(N-methylanthraniloyl)-ATP (mantATP) and ATP of all these mutants except Lys51 were similar. Lys51 mutants were unable to bind mantATP. The pH dependence of V and the V/Ks for MgATP and Fru 6-P suggest a mechanism in which reactants and enzyme combine irrespective of the protonation state of groups required for binding and catalysis, but only the correctly protonated enzyme-substrate complex is catalytically active. A chemical mechanism is suggested in which a general base accepts a proton from the 2-hydroxyl of Fru 6-P concomitant with nucleophilic attack on the gamma-phosphate of MgATP. Phosphoryl transfer is also facilitated by interaction of the gamma-phosphate with a positively charged residue that neutralizes the remaining negative charge. The dianionic form of the 6-phosphate of fructose 6-P is required for binding, and it is likely anchored by a positively charged enzyme residue. A comparison of the pH dependence of kinetic parameters for Ala or His mutant proteins at Lys51, Lys172, and Arg79 suggests that Lys51 interacts with the gamma-phosphate of MgATP and that several other arginines likely participate in transition state stabilization of the transferred phosphoryl. The active site general base has yet to be identified.
Asunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/metabolismo , Animales , Sitios de Unión/genética , Catálisis , Concentración de Iones de Hidrógeno , Cinética , Masculino , Modelos Químicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Fosfofructoquinasa-2 , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Ratas , Testículo/enzimología , ortoaminobenzoatos/metabolismoRESUMEN
To investigate the role in catalysis and/or substrate binding of the Walker motif residues of rat testis fructose 6-phosphate, 2-kinase:fructose-2,6-bisphosphatase (Fru 6-P,2-kinase:Fru-2,6-Pase), we have constructed and characterized mutant enzymes of Asp-128, Thr-52, Asn-73, Thr-130, and Tyr-197. Replacement of Asp-128 by Ala, Asn, and Ser resulted in a small decrease in Vmax and a significant increase in Km values for both substrates. These mutants exhibited similar pH activity profiles as that of the wild type enzyme. Mutation of Thr-52 to Ala resulted in an enzyme with an infinitely high Km for both substrates and an 800-fold decreased Vmax. Substitution of Asn-73 with Ala or Asp caused a 100- and 600-fold increase, respectively in KFru 6-P with only a small increase in KATP and small changes in Vmax. Mutation of Thr-130 caused small changes in the kinetic properties. Replacement of Tyr-197 with Ser resulted in an enzyme with severely decreased binding of Fru 6-P with 3-fold decreased Vmax. A fluorescent analog of ATP, 2'(3')-O-(N-methylanthraniloyl)ATP (mant-ATP) served as a substrate with Km = 0.64 microM, and Vmax = 25 milliunits/mg and was a competitive inhibitor with respect to ATP. When mant-ATP bound to the enzyme, fluorescence intensity at 440 nm increased. mant-ATP binding of the wild type and the mutant enzymes were compared using the fluorometric method. The Kd values of the T52A and D128N enzymes were infinitely high and could not be measured, while those of the other mutant enzymes increased slightly. These results provide evidence that those amino acids are involved in substrate binding, and they are consistent with the crystallographic data. The results also suggest that Asp-128 does not serve as a nucleophile in catalysis, and since there are no other potential nucleophiles in the active site, we hypothesize that the Fru 6-P,2-kinase reaction is mediated via a transition state stabilization mechanism.
Asunto(s)
Aminoácidos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Testículo/enzimología , Animales , Sitios de Unión , Concentración de Iones de Hidrógeno , Cinética , Masculino , Músculos/enzimología , Mutagénesis Sitio-Dirigida , Fosfofructoquinasa-2 , Monoéster Fosfórico Hidrolasas/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Conejos , RatasRESUMEN
BACKGROUND: Glucose homeostasis is maintained by the processes of glycolysis and gluconeogenesis. The importance of these pathways is demonstrated by the severe and life threatening effects observed in various forms of diabetes. The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase catalyzes both the synthesis and degradation of fructose-2,6-bisphosphate, a potent regulator of glycolysis. Thus this bifunctional enzyme plays an indirect yet key role in the regulation of glucose metabolism. RESULTS: We have determined the 2.0 A crystal structure of the rat testis isozyme of this bifunctional enzyme. The enzyme is a homodimer of 55 kDa subunits arranged in a head-to-head fashion, with each monomer consisting of independent kinase and phosphatase domains. The location of ATPgammaS and inorganic phosphate in the kinase and phosphatase domains, respectively, allow us to locate and describe the active sites of both domains. CONCLUSIONS: The kinase domain is clearly related to the superfamily of mononucleotide binding proteins, with a particularly close relationship to the adenylate kinases and the nucleotide-binding portion of the G proteins. This is in disagreement with the broad speculation that this domain would resemble phosphofructokinase. The phosphatase domain is structurally related to a family of proteins which includes the cofactor independent phosphoglycerate mutases and acid phosphatases.
Asunto(s)
Monoéster Fosfórico Hidrolasas/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Proteínas de Unión al GTP/química , Masculino , Datos de Secuencia Molecular , Fosfofructoquinasa-2 , Fosfoglicerato Mutasa/química , Conformación Proteica , Ratas , Testículo/enzimologíaRESUMEN
In order to determine environments around four tryptophan residues, located in the N-terminus, in the kinase and in the phosphatase domains of rat testis Fru 6-P,2-kinase:Fru 2,6-bisphosphatase, mutant enzymes containing a single tryptophan were constructed by site-directed mutagenesis. The kinetic constants of these mutant enzymes were similar to those of the wild-type enzyme. The sum of the fluorescence intensities of the enzymes was 1.5 x that of the wild-type enzyme, and Trp 299, Trp 64, Trp 15, and Trp 320 contributed 38%, 28%, 17%, and 17%, respectively. The fluorescence polarization of the wild-type enzyme was significantly lower than any of the mutant enzymes, suggesting proximity of two tryptophan residues in the wild-type enzyme. The polarization in the presence of Fru 6-P affected only Trp 15, which suggested that it is located near the Fru 6-P binding site, but Trp 64 is not. Inactivation of both enzyme activities and unfolding of these enzymes in guanidine were monitored by activity assays and fluorescence intensities and maxima. Both Fru 6-P,2-kinase and Fru 2,6-bisphosphatase activities of all these enzymes were inactivated between 0.7 and 1 M guanidine. Enzymes containing Trp 64 or Trp 15 showed biphasic fractional unfolding curves, but those of Trp 299 or Trp 320 showed gradual steady changes. Fluorescence quenching by iodide indicated that Trp 64 was not accessible and that other Trp residues were only slightly accessible to solvent. These results suggest that all the Trp residues are in heterogeneous environments and that none are exposed on the protein surface.
Asunto(s)
Complejos Multienzimáticos/química , Monoéster Fosfórico Hidrolasas/química , Fosfotransferasas/química , Testículo/enzimología , Triptófano/química , Animales , Escherichia coli/genética , Polarización de Fluorescencia , Guanidina , Guanidinas/farmacología , Masculino , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Mutagénesis Sitio-Dirigida , Fosfofructoquinasa-2 , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Mutación Puntual , Ratas , Proteínas Recombinantes de Fusión/química , Espectrometría de FluorescenciaRESUMEN
The glucose-stimulated rise in Fru-2,6-P2 in liver results from xylulose 5-P activation of a specific protein phosphatase 2A which dephosphorylates Fru-6-P,2-kinase:Fru-2,6-bisphosphatase (Nishimura, M., and Uyeda, K. (1994) J. Biol. Chem. 269, 26100-26106). In order to determine the role of xylulose 5-P in regulating Fru-2, 6-P2 in liver, the effect of fatty acids, various hexoses, and hormones was examined in perfused rat liver and in intact rats. When 24-h starved rat livers were perfused with acetate, butyrate, or propionate, Fru-2,6-P2 and xylulose 5-P decreased to the same extent and at similar rates. The activity ratios of the kinase and the phosphatase changed in a reciprocal manner, indicating that the phosphorylated form of the enzyme was increased by the fatty acids perfusion. The fatty acids caused the similar changes in the metabolites and the phosphorylation state of the bifunctional enzyme in livers of fed animals. Fructose, galactose, or mannose perfusion in starved rat liver increased both Fru-2,6-P2 and xylulose 5-P and converted the bifunctional enzyme to the dephospho form. Both the Fru-2,6-P2 and xylulose 5-P levels in rats fed a high fat diet decreased over 50% compared to that in control rats. These results indicated a close correlation between Fru-2,6-P2 and xylulose 5-P levels and the phosphorylation state of fructose 6-P, 2-kinase:fructose 2,6-bisphosphatase. Fatty acid inhibition of glucose metabolism can be explained by a decrease in xylulose 5-P, which lowers xylulose 5-P-activated protein phosphatase 2A activity, resulting in more phosphorylated form of the bifunctional enzyme and consequently lower Fru-2,6-P2.
Asunto(s)
Ácidos Grasos/metabolismo , Glucosa/metabolismo , Hígado/metabolismo , Pentosafosfatos/metabolismo , Animales , Carbohidratos de la Dieta/metabolismo , Grasas de la Dieta/metabolismo , Activación Enzimática , Fructosafosfatos/metabolismo , Glucagón/fisiología , Hexosafosfatos/metabolismo , Insulina/fisiología , Masculino , Fosfoproteínas Fosfatasas/metabolismo , Proteína Fosfatasa 2 , Ratas , Ratas Sprague-DawleyRESUMEN
In order to determine the mechanism for delayed increase in Fructose 2,6-P2 in livers of refed rats, the time course of changes in various metabolites upon refeeding NIH or high sucrose diet was investigated. Kinetics of increase in Fructose 2,6-P2 and Xylulose 5-P were similar but different from hexose 6-P or glycogen in the livers of 48 h starved rats refed with NIH diet. The increase in the Fructose 2,6-P2 level was a result of a combination of changes in Fructose 6-P,2-kinase and Fructose 2,6-bisphosphatase activity ratios, indicating dephosphorylation of the bifunctional enzyme and decreased cAMP. A similar correlation between Fructose 2,6-P2 and Xylulose 5-P and dephosphorylation was observed with refeeding high sucrose diet and also with 16 h starved rats. These kinetic results are consistent with the idea that a specific protein phosphatase 2A, activated by Xylulose 5-P, dephosphorylates Fructose 6-P,2-kinase:Fructose 2,6-bisphosphatase and also decreased protein kinase A activity, resulting in increased hepatic Fructose 2,6-P2.
Asunto(s)
AMP Cíclico/metabolismo , Alimentos , Fructosadifosfatos/metabolismo , Hígado/metabolismo , Pentosafosfatos/metabolismo , Animales , Carbohidratos de la Dieta/administración & dosificación , Masculino , Fosfofructoquinasa-2 , Monoéster Fosfórico Hidrolasas/metabolismo , Fosforilación , Ratas , Ratas Sprague-DawleyRESUMEN
Polymerase chain reaction analysis of the mRNA isolated from rat islets demonstrated that the major isozyme of Fructose 6-P,2-kinase:Fructose 2,6-bisphosphatase was the heart type enzyme, and that the liver type enzyme was not detectable. The islet enzyme was expressed in Escherichia coli and purified to homogeneity. The islet enzyme showed the highest Fructose 6-P,2-kinase activity (478 milliunits/mg) compared to the other isozymes and Fructose 2,6-Pase activity (39 milliunits/mg). Fructose 6-P,2-kinase showed KmF6P = 17 microM, which is within the range of in vivo Fru 6-P concentrations in islets. 6-P-Gluconate was a potent inhibitor of Fructose 2,6-Pase. The data suggest that Fructose 6-P,2-kinase activity of the bifunctional enzyme was high and Fructose 2,6-Pase activity was inhibited under physiological variations of blood glucose concentration.
Asunto(s)
Expresión Génica , Islotes Pancreáticos/enzimología , Isoenzimas/metabolismo , Complejos Multienzimáticos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfotransferasas/metabolismo , Animales , Secuencia de Bases , Glucemia , Bovinos , Cartilla de ADN , Isoenzimas/biosíntesis , Isoenzimas/química , Cinética , Hígado/enzimología , Masculino , Datos de Secuencia Molecular , Complejos Multienzimáticos/biosíntesis , Complejos Multienzimáticos/química , Músculo Esquelético/enzimología , Miocardio/enzimología , Especificidad de Órganos , Fosfofructoquinasa-2 , Monoéster Fosfórico Hidrolasas/biosíntesis , Monoéster Fosfórico Hidrolasas/química , Fosfotransferasas/biosíntesis , Fosfotransferasas/química , Reacción en Cadena de la Polimerasa , ARN Mensajero/análisis , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Testículo/enzimologíaRESUMEN
We have shown previously (Nishimura, M., Fedorov, S., and Uyeda, K. (1994) (J. Biol. Chem. 269, 26100-26106) that the administration of high concentrations of glucose stimulates dephosphorylation of Fru-6-P,2-kinase: Fru-2,6-bisphosphatase in perfused liver, and xylulose (Xu) 5-P activates the dephosphorylation reaction. To characterize the protein phosphatase, we have purified the Xu 5-P-activated protein phosphatase to homogeneity from livers of rats injected with high glucose. Several protein phosphatases in the livers were separated by DEAE-cellulose chromatography, but only one peak of the enzyme was activated by Xu 5-P. The protein phosphatase was inhibited by okadaic acid (IC50 = 1-3 nM) and did not require Mg2+ or Ca2+, suggesting that the enzyme was type 2A. The enzyme was a heterotrimer (M(r) = 150,000) and consisted of structural (A, 65 kDa) catalytic (C, 36 kDa), and regulatory (B, 52 kDa) subunits. Amino acid sequences of five tryptic peptides derived from the B subunit showed similarity with those of the B alpha isoform of rat protein phosphatase 2A, but five out of 73 residues were different. The protein phosphatase catalyzed dephosphorylation of Fru-6-P,2-kinase:Fru-2,6-Pase, phosphorylase alpha, and pyruvate kinase, and the Km values were 0.8 microM, 3.7 microM, and 2.2 microM, respectively. Among these substrates dephosphorylation of only the bifunctional enzyme was activated by Xu 5-P, and the K alpha value for Xu 5-P was 20 microM. Xu 5-P was the only sugar phosphate which activated the PP2A among all the sugar phosphates examined. These results demonstrated the existence and isolation of a unique heterotrimeric protein phosphatase 2A in rat liver which catalyzed the dephosphorylation of Fru-6-P,2-kinase:Fru-2,6-Pase and was activated specifically by Xu 5-P. The Xu 5-P-activated protein phosphatase 2A explains the increased Fru 2,6-P2 level in liver after high glucose administration.
Asunto(s)
Hígado/enzimología , Complejos Multienzimáticos/metabolismo , Pentosafosfatos/farmacología , Fosfoproteínas Fosfatasas/aislamiento & purificación , Fosfoproteínas Fosfatasas/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Fosfotransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Cromatografía de Afinidad , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Éteres Cíclicos/farmacología , Sustancias Macromoleculares , Masculino , Datos de Secuencia Molecular , Peso Molecular , Ácido Ocadaico , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Fosfofructoquinasa-2 , Fosfoproteínas Fosfatasas/química , Proteína Fosfatasa 2 , Ratas , Ratas Sprague-Dawley , Especificidad por Sustrato , Testículo/enzimologíaRESUMEN
Diffraction-quality crystals of the bifunctional enzyme fructose 6-phosphate, 2-kinase:fructose 2,6-bisphosphatase from rat testis have been obtained. The crystals were grown in the presence of ATP gamma S, fructose 6-phosphate, the detergent n-octylglucoside, and the precipitant polyethylene glycol 4000. The crystals have the symmetry of the trigonal space group P31/221 with a = b = 83.0 A and c = 130.6 A. Flash-frozen crystals diffract to beyond 2.2 A, and native data have been collected.