RESUMEN

1. Multidrug and toxic compound extrusion (MATE)-type transporters, which were first identified as a bacterial drug transporter family, are present in almost all prokaryotes and eukaryotes, and are thus one of the mostly conserved transporter families in nature. 2. Recently, a mammalian MATE transporter was shown to be a long hypothesized electroneutral H(+)/organic cation exporter that is responsible for the excretion of metabolic waste products and xenobiotics at renal brush border membranes and bile canaliculi. Plant MATE-type transporters are involved in the detoxification of metals and secondary metabolites such as phenols through their vesicular storage or extrusion at the plasma membrane. 3. Thus, MATE transporters are involved in one of the basic mechanisms that maintain homeostasis through the excretion of metabolic waste products and xenobiotics in nature.

Asunto(s)

Antiportadores/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Xenobióticos/metabolismo , Animales , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Canalículos Biliares/metabolismo , Transporte Biológico/fisiología , Resistencia a Múltiples Medicamentos/fisiología , Humanos , Metales/metabolismo , Microvellosidades/metabolismo , Fenoles/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo

RESUMEN

Coupling with electrochemical proton gradient, ATP synthase (F(0)F(1)) synthesizes ATP from ADP and phosphate. Mutational studies on high-resolution structure have been useful in understanding this complicated membrane enzyme. We discuss mainly the mechanism of catalysis in the beta subunit of F(1) sector and roles of the gamma subunit in energy coupling. The gamma-subunit rotation during catalysis is also discussed.

Asunto(s)

ATPasas de Translocación de Protón/química , Bacterias/enzimología , Sitios de Unión , Cloroplastos/enzimología , Cinética , Modelos Moleculares , Proteínas Motoras Moleculares , Mutación , Nucleótidos/metabolismo , Unión Proteica , ATPasas de Translocación de Protón/metabolismo , Protones

RESUMEN

The three catalytic sites of the F(O)F(1) ATP synthase interact through a cooperative mechanism that is required for the promotion of catalysis. Replacement of the conserved alpha subunit Arg-376 in the Escherichia coli F(1) catalytic site with Ala or Lys resulted in turnover rates of ATP hydrolysis that were 2 x 10(3)-fold lower than that of the wild type. Mutant enzymes catalyzed hydrolysis at a single site with kinetics similar to that of the wild type; however, addition of excess ATP did not chase bound ATP, ADP, or Pi from the catalytic site, indicating that binding of ATP to the second and third sites failed to promote release of products from the first site. Direct monitoring of nucleotide binding in the alphaR376A and alphaR376K mutant F(1) by a tryptophan in place of betaTyr-331 (Weber et al. (1993) J. Biol. Chem. 268, 20126-20133) showed that the catalytic sites of the mutant enzymes, like the wild type, have different affinities and therefore, are structurally asymmetric. These results indicate that alphaArg-376, which is close to the beta- or gamma-phosphate group of bound ADP or ATP, respectively, does not make a significant contribution to the catalytic reaction, but coordination of the arginine to nucleotide filling the low-affinity sites is essential for promotion of rotational catalysis to steady-state turnover.

Asunto(s)

Arginina/metabolismo , Dominio Catalítico , Escherichia coli/enzimología , ATPasas de Translocación de Protón/metabolismo , Sustitución de Aminoácidos/genética , Arginina/genética , Sitios de Unión/genética , Catálisis , Dominio Catalítico/genética , Escherichia coli/genética , Hidrólisis , Cinética , Mutagénesis Insercional , Mutagénesis Sitio-Dirigida , Fosfatos/metabolismo , ATPasas de Translocación de Protón/biosíntesis , ATPasas de Translocación de Protón/genética

RESUMEN

Utilizing human P-glycoprotein (P-gp), we investigated methods to enhance the heterologous expression of ATP-binding cassette transporters in Saccharomyces cerevisiae. Human multidrug resistance gene MDR1 cDNA was placed in a high-copy 2 mu yeast expression plasmid under the control of the inducible GAL1 promoter or the strong constitutive PMA1 promoter from which P-gp was expressed in functional form. Yeast cells expressing P-gp were valinomycin resistant. Basal ATPase activity of P-gp in yeast membranes was 0. 4-0.7 micromol/mg/min indicating excellent functionality. P-glycoprotein expressed in the protease-deficient strain BJ5457 was found in the plasma membrane and was not N-glycosylated. By use of the PMA1 promoter, P-gp could be expressed at 3% of total membrane protein. The expression level could be further enhanced to 8% when cells were grown in the presence of 10% glycerol as a chemical chaperone. Similarly, glycerol enhanced protein levels of P-gp expressed under control of the GAL1 promoter. Glycerol was demonstrated to enhance posttranslational stability of P-gp. Polyhistidine-tagged P-gp was purified by metal affinity chromatography and reconstituted into proteoliposomes in milligram quantities and its ATPase activity was characterized. Turnover numbers as high as 12 s(-1) were observed. The kinetic parameters K(MgATP)(M), V(max), and drug activation were dependent on the lipid composition of proteoliposomes and pH of the assay and were similar to P-gp purified from mammalian sources. In conclusion, we developed a system for cost-effective, high-yield, heterologous expression of functional P-gp useful in producing large quantities of normal and mutant P-gp forms for structural and mechanistic studies.

Asunto(s)

Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/aislamiento & purificación , Secuencia de Bases , Membrana Celular/metabolismo , Cartilla de ADN/genética , Expresión Génica/efectos de los fármacos , Vectores Genéticos , Glicerol/farmacología , Humanos , Técnicas In Vitro , Ionóforos/farmacología , Cinética , Monensina/farmacología , Mutación , Plásmidos/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Saccharomyces cerevisiae/efectos de los fármacos

RESUMEN

Arabidopsis thaliana vacuolar H(+)-translocating pyrophosphatase (V-PPase) was expressed functionally in yeast vacuoles with endogenous vacuolar H(+)-ATPase (V-ATPase), and the regulation and reversibility of V-ATPase were studied using these vacuoles. Analysis of electrochemical proton gradient (DeltamuH) formation with ATP and pyrophosphate indicated that the proton transport by V-ATPase or V-PPase is not regulated strictly by the proton chemical gradient (DeltapH). On the other hand, vacuolar membranes may have a regulatory mechanism for maintaining a constant membrane potential (DeltaPsi). Chimeric vacuolar membranes showed ATP synthesis coupled with DeltamuH established by V-PPase. The ATP synthesis was sensitive to bafilomycin A(1) and exhibited two apparent K(m) values for ADP. These results indicate that V-ATPase is a reversible enzyme. The ATP synthesis was not observed in the presence of nigericin, which dissipates DeltapH but not DeltaPsi, suggesting that DeltapH is essential for ATP synthesis.

Asunto(s)

Adenosina Trifosfato/metabolismo , Macrólidos , ATPasas de Translocación de Protón/metabolismo , Pirofosfatasas/metabolismo , ATPasas de Translocación de Protón Vacuolares , Vacuolas/enzimología , Antibacterianos/farmacología , Arabidopsis/enzimología , Transporte Biológico , Compartimento Celular , Regulación Enzimológica de la Expresión Génica , Membranas Intracelulares/enzimología , Fuerza Protón-Motriz , ATPasas de Translocación de Protón/antagonistas & inhibidores , ATPasas de Translocación de Protón/genética , Pirofosfatasas/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética

RESUMEN

F0F1, found in mitochondria or bacterial membranes, synthesizes adenosine 5'-triphosphate (ATP) coupling with an electrochemical proton gradient and also reversibly hydrolyzes ATP to form the gradient. An actin filament connected to a c subunit oligomer of F0 was able to rotate by using the energy of ATP hydrolysis. The rotary torque produced by the c subunit oligomer reached about 40 piconewton-nanometers, which is similar to that generated by the gamma subunit in the F1 motor. These results suggest that the gamma and c subunits rotate together during ATP hydrolysis and synthesis. Thus, coupled rotation may be essential for energy coupling between proton transport through F0 and ATP hydrolysis or synthesis in F1.

Asunto(s)

Adenosina Trifosfato/metabolismo , Proteínas Motoras Moleculares/química , Proteínas Motoras Moleculares/metabolismo , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/metabolismo , Actinas/química , Actinas/metabolismo , Sitios de Unión , Biotinilación , Transferencia de Energía , Enzimas Inmovilizadas , Escherichia coli/enzimología , Hidrólisis , Fuerza Protón-Motriz , Desacopladores/metabolismo , Desacopladores/farmacología , Venturicidinas/farmacología , Grabación en Video

RESUMEN

The rotation of the gamma-subunit has been included in the binding-change mechanism of ATP synthesis/hydrolysis by the proton ATP synthase (FOF1). The Escherichia coli ATP synthase was engineered for rotation studies such that its ATP hydrolysis and synthesis activity is similar to that of wild type. A fluorescently labeled actin filament connected to the gamma-subunit of the F1 sector rotated on addition of ATP. This progress enabled us to analyze the gammaM23K (the gamma-subunit Met-23 replaced by Lys) mutant, which is defective in energy coupling between catalysis and proton translocation. We found that the F1 sector produced essentially the same frictional torque, regardless of the mutation. These results suggest that the gammaM23K mutant is defective in the transformation of the mechanical work into proton translocation or vice versa.

Asunto(s)

Actinas/química , Escherichia coli/enzimología , Escherichia coli/genética , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/metabolismo , Sustitución de Aminoácidos , Cinética , Sustancias Macromoleculares , Mutagénesis Insercional , Operón , ATPasas de Translocación de Protón/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rotación , Torque

RESUMEN

The role of the conserved sequence motif 301DDLTDP306 in the F0F1 ATP synthase beta subunit was assessed by mutagenic analysis in the Escherichia coli enzyme. Mutations gave variable effects on F1 sector activity, stability, and membrane binding to the F0 sector. Upon solubilization, F1 sectors of the betaD302E and betaD305E mutants (betaAsp-302 and betaAsp-305 replaced by glutamate) dissociated into subunits, while mutants with other beta305 substitutions failed to assemble. Membrane ATPase activities of beta301 and 302 mutants were 20-70% of wild type. Replacements of the gamma subunit Gln-269 had similar effects. The membrane ATPase activities of the gammaQ269E or gammaQ269D mutants were significantly lower and their F1 sectors dissociated into subunits upon solubilization. These results suggest that the beta301-305 loop and the gamma subunit region around Gln-269 form a key region for the assembly of alpha3 beta3 gamma complex. These results are consistent with the X-ray crystallographic structure of bovine F1 (J. P. Abrahams, A. G. W. Leslie, R. Lutter, and J. E. Walker (1994) Nature 370, 621-628) where the beta301DDLTD305 loop directly interacts with gammaGln-269.

Asunto(s)

Sustitución de Aminoácidos/genética , Ácido Aspártico/metabolismo , Glutamina/metabolismo , ATPasas de Translocación de Protón/metabolismo , Ácido Aspártico/genética , Activación Enzimática/genética , Estabilidad de Enzimas/genética , Escherichia coli/enzimología , Escherichia coli/genética , Mutación del Sistema de Lectura , Glutamina/genética , Membranas Intracelulares/enzimología , Mutagénesis Sitio-Dirigida , Fosforilación Oxidativa , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , ATPasas de Translocación de Protón/genética

RESUMEN

Escherichia coli ATP synthase has eight subunits and functions through transmission of conformational changes between subunits. Extensive mutational analyses identified essential residues for catalysis and conformation transmission. Pseudorevertant studies revealed that beta/alpha and beta/gamma subunits interactions are important for the energy coupling between catalysis and H+ translocation. In this article, we discuss mechanism of catalysis and energy coupling based on our recent mutation studies.

Asunto(s)

Análisis Mutacional de ADN , ATPasas de Translocación de Protón Mitocondriales , ATPasas de Translocación de Protón/genética , ATPasas de Translocación de Protón/metabolismo , Proteínas de Saccharomyces cerevisiae , Secuencia de Aminoácidos/genética , Catálisis , Metabolismo Energético/fisiología , Escherichia coli/enzimología , Modelos Moleculares , Mutación/fisiología

RESUMEN

Caenorhabditis elegans putative copper ATPase (CUA-1) had been functionally expressed in a yeast delta ccc2 mutant (copper ATPase gene disruptant). We found that CUA-1 with Cys-Pro-Cys to Cys-Pro-Ala mutation could not rescue the yeast delta ccc2 mutant, suggesting that the carboxyl terminal cysteine residue in the conserved Cys-Pro-Cys motif is essential for copper transport.

Asunto(s)

Adenosina Trifosfatasas/química , Caenorhabditis elegans/enzimología , Cobre/farmacocinética , Oligopéptidos/análisis , Secuencia de Aminoácidos , Animales , Transporte Biológico , Datos de Secuencia Molecular , Mutación , Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido

Asunto(s)

Escherichia coli/enzimología , Estructura Secundaria de Proteína , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/ultraestructura , Membrana Celular/enzimología , Liposomas , Sustancias Macromoleculares , Microscopía de Fuerza Atómica/métodos , Modelos Moleculares

RESUMEN

The full-length cDNA coding for a putative copper transporting P-type ATPase (Cu2+-ATPase) was cloned from Caenorhabditis elegans. The putative Cu2+-ATPase is a 1,238-amino acid protein, and highly homologous to the Menkes and Wilson disease gene products mutations of which are responsible for human defects of copper metabolism. The Saccharomyces cerevisiae mutant with a disrupted CCC2 gene (yeast Menkes/Wilson disease gene homologue) was rescued by the cDNA for the C. elegans Cu2+-ATPase but not by the cDNA with an Asp-786 (an invariant phosphorylation site) to Asn mutation, suggesting that the C. elegans Cu2+-ATPase functions as a copper transporter in yeast. The expressed C. elegans protein was detected in yeast vacuolar membranes by immunofluorescence microscopy. The yeast expression system may facilitate further studies on copper transporting P-type ATPases.

Asunto(s)

Caenorhabditis elegans/genética , Eliminación de Gen , Genes Fúngicos , Degeneración Hepatolenticular/genética , Síndrome del Pelo Ensortijado/genética , Saccharomyces cerevisiae/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/genética , Humanos , Datos de Secuencia Molecular , Mutación , Homología de Secuencia de Ácido Nucleico

RESUMEN

Escherichia coli ATP synthase has eight subunits and functions through transmission of conformational changes between subunits. Defective mutation at beta Gly-149 was suppressed by the second mutations at the outer surface of the beta subunit, indicating that the defect by the first mutation was suppressed by the second mutation through long range conformation transmission. Extensive mutant/pseudorevertant studies revealed that beta/alpha and beta/gamma subunits interactions are important for the energy coupling between catalysis and H+ translocation. In addition, long range interaction between amino and carboxyl terminal regions of the gamma subunit has a critical role(s) for energy coupling. These results suggest that the dynamic conformation change and its transmission are essential for ATP synthase.

Asunto(s)

Conformación Proteica , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/metabolismo , Catálisis , Escherichia coli/enzimología , Escherichia coli/genética , Mutagénesis , ATPasas de Translocación de Protón/genética , Relación Estructura-Actividad

RESUMEN

The structure of Escherichia coli F0F1-ATPase (ATP synthase), and its F0 sector reconstituted in lipid membranes was analyzed using atomic force microscopy (AFM) by tapping-mode operation. The majority of F0F1-ATPases were visualized as spheres with a calculated diameter of approximately 90 angstroms, and a height of approximately 100 angstroms from the membrane surface. F0 sectors were visualized as two different ring-like structures (one with a central mass and the other with a central hollow of greater than or equal to 18 angstroms depth) with a calculated outer diameter of approximately 130 angstroms. The two different images possibly represent the opposite orientations of the complex in the membranes. The ring-like projections of both images suggest inherently asymmetric assemblies of the subunits in the F0 sector. Considering the stoichiometry of F0 subunits, the area of the image observed is large enough to accommodate all three F0 subunits in an asymmetric manner.

Asunto(s)

Escherichia coli/enzimología , Proteínas de la Membrana/química , ATPasas de Translocación de Protón/química , Membrana Celular/enzimología , Microscopía de Fuerza Atómica

Asunto(s)

Escherichia coli/enzimología , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/metabolismo , Secuencia de Aminoácidos , Sitios de Unión/genética , Catálisis , Metabolismo Energético , Escherichia coli/genética , Hidrólisis , Modelos Químicos , Datos de Secuencia Molecular , Mutación , Conformación Proteica , ATPasas de Translocación de Protón/genética

RESUMEN

Glu-beta 185 of the Escherichia coli H(+)-ATPase (ATP synthase) beta subunit was replaced by 19 different amino acid residues. The rates of multisite (steady state) catalysis of all the mutant membrane ATPases except Asp- beta 185 were less than 0.2% of the wild type one; the Asp- beta 185 enzyme exhibited 15% (purified) and 16% (membrane-bound) ATPase activity. The purified inactive Cys- beta 185 F1-ATPase recovered substantial activity after treatment with iodoacetate in the presence of MgCl2; maximal activity was obtained upon the introduction of about 3 mol of carboxymethyl residues/mol of F1. The divalent cation dependences of the S-carboxymethyl- beta 185 and Asp- beta 185 ATPase activities were altered from that of the wild type. The Asp- beta 185, Cys- beta 185, S-carboxymethyl-beta 185, and Gln- beta 185 enzymes showed about 130, 60, 20, and 50% of the wild type unisite catalysis rates, respectively. The S-carboxymethyl- beta 185 and Asp- beta 185 enzymes showed altered divalent cation sensitivities, and the S-carboxymethyl- beta 185 enzyme showed no Mg2+ inhibition. Unlike the wild type, the two mutant enzymes showed low sensitivities to azide, which stabilizes the enzyme Mg-ADP complex. These results suggest that Glu- beta 185 may form a Mg2+ binding site, and its carboxyl moiety is essential for catalytic cooperativity. Consistent with this model, the bovine glutamate residue corresponding to Glu- beta 185 is located close to the catalytic site in the higher order structure (Abrahams, J.P., Leslie, A.G.W., Lutter, R ., and Walker, J.E. (1994) Nature 370, 621-628)

Asunto(s)

Escherichia coli/enzimología , ATPasas de Translocación de Protón/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Azidas/farmacología , Sitios de Unión , Secuencia Conservada , Relación Dosis-Respuesta a Droga , Activación Enzimática , Escherichia coli/genética , Ácido Glutámico/genética , Yodoacetatos/farmacología , Ácido Yodoacético , Cloruro de Litio/farmacología , Cloruro de Magnesio/farmacología , Metilación , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , ATPasas de Translocación de Protón/efectos de los fármacos , ATPasas de Translocación de Protón/genética , Relación Estructura-Actividad

RESUMEN

The mechanisms of energy coupling and catalytic co-operativity are not yet understood for H(+)-ATPase (ATP synthase). An Escherichia coli gamma subunit frameshift mutant (downstream of Thr-gamma 277) could not grow by oxidative phosphorylation because both mechanisms were defective (Iwamoto, A., Miki, J., Maeda, M., and Futai, M. (1990) J. Biol. Chem. 265, 5043-5048). The defect(s) of the gamma frameshift was obvious, because the mutant subunit had a carboxyl terminus comprising 16 residues different from those in the wild type. However, in this study, we surprisingly found that an Arg-beta 52-->Cys or Gly-beta 150-->Asp replacement could suppress the deleterious effects of the gamma frameshift. The membranes of the two mutants (gamma frameshift/Cys-beta 52 with or without a third mutation, Val-beta 77-->Ala) exhibited increased oxidative phosphorylation, together with 70-100% of the wild type ATPase activity. Similarly, the gamma frameshift/Asp-beta 150 mutant could grow by oxidative phosphorylation, although this mutant had low membrane ATPase activity. These results suggest that the beta subunit mutation suppressed the defects of catalytic cooperativity and/or energy coupling in the gamma mutant, consistent with the notion that conformational transmission between the two subunits is pertinent for this enzyme.

Asunto(s)

Escherichia coli/enzimología , Mutación Puntual , Estructura Secundaria de Proteína , ATPasas de Translocación de Protón/química , ATPasas de Translocación de Protón/metabolismo , Secuencia de Aminoácidos , Arginina , Ácido Aspártico , Secuencia de Bases , Sitios de Unión , Catálisis , Cisteína , Cartilla de ADN , Escherichia coli/genética , Mutación del Sistema de Lectura , Glicina , Cinética , Sustancias Macromoleculares , Modelos Estructurales , Datos de Secuencia Molecular , Mutagénesis , Operón , Fosforilación Oxidativa , Plásmidos , Reacción en Cadena de la Polimerasa , ATPasas de Translocación de Protón/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo

RESUMEN

Twenty-two mutants between beta Glu-161 and beta Lys-201 of Escherichia coli H(+)-ATPase beta subunit could grow by oxidative phosphorylation, but 11 other such mutants, beta Glu-181-->Gln, Asp, Asn, Thr, Ser, Ala, or Lys and beta Arg-182-->Lys, Ala, Glu, or Gln, could not. The beta Asp-181, beta Lys-182, and other defective mutants had 1.4, 1, and < 0.1%, respectively, of the wild-type membrane ATPase activity. Partially purified F1-ATPases from all mutants at positions 181 and 182, except for the beta Asp-181 and beta Lys-182 mutants, showed very low unisite catalysis. Purified F1-ATPases of the beta Gln-181 and beta Ala-181 mutants showed no multisite (or steady state) catalysis and slow unisite catalysis (< or = 1% of that of the wild type): their defects could be attributed to decreased catalytic rates (low k+2 and k-2). Changes of the k+2 and k-2 values in the beta Asp-181 enzyme, which showed detectable multi- and unisite catalysis, were less marked (27 and 21%, respectively, of wild-type rates). The beta Gln-182 enzyme showed defective catalysis (< or = 0.1% of the multi- and approximately 1% of the unisite catalyses of the wild type), whereas the beta Lys-182 enzyme showed 1 and 85% of the wild-type multisite and unisite catalytic rates, respectively. beta Lys-182 had wild-type values of k+2 and k-2, but beta Gln-182 had k+2 about 10-fold lower than that of wild type.(ABSTRACT TRUNCATED AT 250 WORDS)

Asunto(s)

Arginina/química , Ácido Glutámico/química , ATPasas de Translocación de Protón/química , Nucleótidos de Adenina , Secuencia de Bases , Sitios de Unión , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida

RESUMEN

The molecular biological approach has provided important information for understanding the F0F1 H(+)-ATPase. This article focuses on our recent results on the catalytic site in the beta subunit, and the roles of alpha/beta subunit interaction and amino/carboxyl terminal interaction of the gamma subunit in energy coupling. Extensive mutagenesis of the beta subunit revealed that beta Lys-155, beta Thr-156, beta Glu-181 and beta Arg-182 are essential catalytic residues. beta Glu-185 is not absolutely essential, but a carboxyl residue may be necessary at this position. A pseudo-revertant analysis positioned beta Gly-172, beta Ser-174, beta Glu-192 and beta Val-198 in the proximity of beta Gly-149. The finding of the roles of beta Gly-149, beta Lys-155, and beta Thr-156 emphasized the importance of the glycine-rich sequence (Gly-X-X-X-X-Gly-Lys-Thr/Ser, E. coli beta residues between beta Gly-149 and beta Thr-156) conserved in many nucleotide binding proteins. The A subunits of vacuolar type ATPases may have a similar catalytic mechanism because they have conserved glycine-rich and Gly-Glu-Arg (corresponding to beta Gly-180-beta Arg-182) sequences. The results of these mutational studies are consistent with the labeling of beta Lys-155 and beta Lys-201 with AP3-PL, and of beta Glu-192 with DCCD [15]. The DCCD-binding residue of a thermophilic Bacillus corresponds to beta Glu-181, an essential catalytic residue discussed above. The defective coupling of the beta Ser-174-->Phe mutant was suppressed by the second mutation alpha Arg-296-->Cys, indicating the importance of alpha/beta interaction in energy coupling. The gamma subunit, especially its amino/carboxyl interaction, seems to be essential for energy coupling between catalysis and transport judging from studies on gamma Met-23-->Lys or Arg mutation and second-site mutations which suppressed the gamma Lys-23 mutation. Thus the conserved gamma Met-23 is not absolutely essential but is located in the important region for amino/carboxyl interaction for energy coupling.

Asunto(s)

Metabolismo Energético , ATPasas de Translocación de Protón/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Escherichia coli , Datos de Secuencia Molecular , Mutación , ATPasas de Translocación de Protón/genética , Alineación de Secuencia

Asunto(s)

ATPasas de Translocación de Protón/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Catálisis , Escherichia coli/enzimología , Glicina , Hidrógeno/metabolismo , Datos de Secuencia Molecular , ATPasas de Translocación de Protón/química