研究者詳細 - 表　弘志

論文 - 表　弘志

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3SD03 P-糖タンパク質の構造と薬剤排出の分子機構(構造から見た膜輸送)

表弘志, Al-Shawi Marwan K.

生物物理 44 S24 2004年

Transition state analysis of the coupling of drug transport to ATP hydrolysis by P-glycoprotein 査読

MK Al-Shawi, MK Polar, H Omote, RA Figler

JOURNAL OF BIOLOGICAL CHEMISTRY 278 ( 52 ) 52629 - 52640 2003年12月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

ATPase activity associated with P-glycoprotein (Pgp) is characterized by three drug-dependent phases: basal (no drug), drug-activated, and drug-inhibited. To understand the communication between drug-binding sites and ATP hydrolytic sites, we performed steady-state thermodynamic analyses of ATP hydrolysis in the presence and absence of transport substrates. We used purified human Pgp (ABCB1, MDR1) expressed in Saccharomyces cerevisiae (Figler, R. A., Omote, H., Nakamoto, R. K., and Al-Shawi, M. K. (2000) Arch. Biochem. Biophys. 376, 34-46) as well as Chinese hamster Pgp (PGP1). Between 23 and 35 degreesC, we obtained linear Arrhenius relationships for the turnover rate of hydrolysis of saturating MgATP in the presence of saturating drug concentrations (k(cat)), from which we calculated the intrinsic enthalpic, entropic, and free energy terms for the rate-limiting transition states. Linearity of the Arrhenius plots indicated that the same rate-limiting step was being measured over the temperature range employed. Using linear free energy analysis, two distinct transition states were found: one associated with uncoupled basal activity and the other with coupled drug transport activity. We concluded that basal ATPase activity associated with Pgp is not a consequence of transport of an endogenous lipid or other endogenous substrates. Rather, it is an intrinsic mechanistic property of the enzyme. We also found that rapidly transported substrates bound tighter to the transition state and required fewer conformational alterations by the enzyme to achieve the coupling transition state. The overall rate-limiting step of Pgp during transport is a carrier reorientation step. Furthermore, Pgp is optimized to transport drugs out of cells at high rates at the expense of coupling efficiency. The drug inhibition phase was associated with low affinity drug-binding sites. These results are consistent with an expanded version of the alternating catalytic site drug transport model (Senior, A. E., Al-Shawi, M. K., and Urbatsch, I. L. (1995) FEBS Lett. 377, 285-289). A new kinetic model of drug transport is presented.

DOI： 10.1074/jbc.M308175200

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A novel electron paramagnetic resonance approach to determine the mechanism of drug transport by P-glycoprotein 査読

H Omote, MK Al-Shawi

JOURNAL OF BIOLOGICAL CHEMISTRY 277 ( 47 ) 45688 - 45694 2002年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

ATP-driven pumping of a variety of drugs out of cells by the human P-glycoprotein poses a serious problem to medical therapy. High level heterologous expression of human P-glycoprotein, in the yeast Saccharomyces cerevisiae, has facilitated biophysical studies in purified proteoliposome preparations. Membrane permeability of transported drugs and consequent lack of an experimentally defined drug position have made resolution of the transport mechanism difficult by classical techniques. To overcome these obstacles we devised a novel EPR spin-labeled verapamil for use as a transport substrate. Spin-labeled verapamil was an excellent transport substrate with apparent turnover number, K-m and K-i values of 5.8 s(-1), 4 mum, and 210 muM, respectively, at pH 7.4 and 37 degreesC. The apparent affinities were similar to10-fold higher than for unlabeled verapamil. Spin-labeled verapamil stimulated ATPase activity similar to5-fold, was relatively hydrophilic, and had a very low flip-flop rate, making it an ideal transport substrate. The K-m for MgATP activation of transport was 0.8 mm. By measuring the mobility of spin-labeled verapamil during transport experiments, we were able to resolve the location of the drug in proteoliposome suspensions. Steady state gradients of spin-labeled verapamil within the range of K-i/K-m ratios were observed.

DOI： 10.1074/jbc.M206479200

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Use of chemical chaperones in the yeast Saccharomyces cerevisiae to enhance heterologous membrane protein expression: High-yield expression and purification of human P-glycoprotein 査読

RA Figler, H Omote, RK Nakamoto, MK Al-Shawi

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 376 ( 1 ) 34 - 46 2000年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE INC

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 mu mol/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-M(MgATP),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. (C) 2000 Academic Press.

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Mechanical rotation of the c subunit oligomer in ATP synthase (F0F1): direct observation. 査読

Sambongi Y, Iko Y, Tanabe M, Omote H, Iwamoto-Kihara A, Ueda I, Yanagida T, Wada Y, Futai M

Science (New York, N.Y.) 286 ( 5445 ) 1722 - 1724 1999年11月

Stability of the Escherichia coli ATP synthase F0F1 complex is dependent on interactions between gamma Gln-269 and the beta subunit loop beta Asp-301-beta Asp-305 査読

H Omote, K Tainaka, K Fujie, A Iwamoto-Kihara, Y Wada, M Futai

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 358 ( 2 ) 277 - 282 1998年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ACADEMIC PRESS INC

The role of the conserved sequence motif (DDLTDP306)-D-301 in the F0F1 ATP synthase beta subunit was assessed by mutagenic analysis in the Escherichia coli enzyme. Mutations gave variable effects on F-1 sector activity, stability, and membrane binding to the F-0 sector. Upon solubilization, F-1 sectors of the beta D302E and beta D305E mutants (beta Asp-302 and beta Asp-305 replaced by glutamate) dissociated into subunits, while mutants with other beta 305 substitutions failed to assemble. Membrane ATPase activities of beta 301 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 gamma Q269E or gamma Q269D mutants were significantly lower and their F-1 sectors dissociated into subunits upon solubilization. These results suggest that the beta 301-305 loop and the gamma subunit region around Gln-269 form a key region for the assembly of alpha(3)beta(3)gamma complex. These results are consistent with the X-ray crystallographic structure of bovine F-1 (J. P. Abrahams, A. G. W. Leslie, R. Lutter, and J. E. Walker (1994) Nature 370, 621-628) where the beta(301)DDLTD(305) loop directly interacts with gamma Gln-269. (C) 1988 Academic Press.

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Essential Cys-Pro-Cys Motif of Caenorhabditis elegans Copper Transport ATPase

Yoshimizu Takao, Omote Hiroshi, Wakabayashi Tokumitsu, SAMBONGI Yoshihiro, FUTAI Masamitsu

Bioscience, biotechnology, and biochemistry 62 ( 6 ) 1258 - 1260 1998年6月

記述言語：英語出版者・発行元：社団法人日本農芸化学会

Caenorhabditis elegans putative copper ATPase (CUA-1) had been functionally expressed in a yeast &lrtri;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 &lrtri;ccc2 mutant, suggesting that the carboxyl terminal cysteine residue in the conserved Cys-Pro-Cys motif is essential for copper transport.

DOI： 10.1271/bbb.62.1258

CiNii Article

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その他リンク： https://jlc.jst.go.jp/DN/JALC/00085042551?from=CiNii

Mutational analysis of F₁F₀ ATPase: Catalysis and energy coupling

Omote, H., Futai, M.

Acta Physiologica Scandinavica, Supplement 163 ( 643 ) 1998年

Atomic force microscopy of Escherichia coli FoF1-ATPase in reconstituted membranes 査読

Takeyasu, K., Omote, H., Nettikadan, S., Tokumasu, F., Iwamoto-Kihara, A., Futai, M.

Annals of the New York Academy of Sciences 834 ( 1 Na/K-ATPase a ) 149 - 152 1997年11月

Conformational transmission in ATP synthase during catalysis: Search for large structural changes

Futai, M., Omote, H.

Journal of Bioenergetics and Biomembranes 28 ( 5 ) 1996年

Molecular imaging of Escherichia coli F₀F₁-ATPase in reconstituted membranes using atomic force microscopy

Takeyasu, K., Omote, H., Nettikadan, S., Tokumasu, F., Iwamoto-Kihara, A., Futai, M.

FEBS Letters 392 ( 2 ) 1996年

Chapter 3 F-type H⁺ ATPase (ATP synthase): Catalytic site and energy coupling

Futal, M., Omote, H.

Handbook of Biological Physics 2 ( C ) 1996年

Escherichia coli H+-ATPase (ATP synthase): Catalytic site and roles of subunit interactions in energy coupling 査読

M Futai, H Omote, M Maeda

BIOCHEMICAL SOCIETY TRANSACTIONS 23 ( 4 ) 785 - 789 1995年11月

BETA-SUBUNIT GLU-185 OF ESCHERICHIA-COLI H+-ATPASE (ATP SYNTHASE) IS AN ESSENTIAL RESIDUE FOR COOPERATIVE CATALYSIS 査読

H OMOTE, NP LE, MY PARK, M MAEDA, M FUTAI

JOURNAL OF BIOLOGICAL CHEMISTRY 270 ( 43 ) 25656 - 25660 1995年10月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

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 F-1-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 F-1. The divalent cation dependences of the S-carboxymethyl-beta 185 and Asp-beta 185 ATPase activities were altered from that of the mild 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 mild 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).

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BETA-GAMMA SUBUNIT INTERACTION IS REQUIRED FOR CATALYSIS BY H+-ATPASE (ATP SYNTHASE) - BETA-SUBUNIT AMINO-ACID REPLACEMENTS SUPPRESS A GAMMA-SUBUNIT MUTATION HAVING A LONG UNRELATED CARBOXYL-TERMINUS 査読

C JEANTEURDEBEUKELAER, H OMOTE, A IWAMOTOKIHARA, M MAEDA, M FUTAI

JOURNAL OF BIOLOGICAL CHEMISTRY 270 ( 39 ) 22850 - 22854 1995年9月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

The mechanisms of energy coupling and catalytic cooperativity 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.

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CONSERVED GLU-181 AND ARG-182 RESIDUES OF ESCHERICHIA-COLI H+-ATPASE (ATP SYNTHASE) BETA-SUBUNIT ARE ESSENTIAL FOR CATALYSIS - PROPERTIES OF 33 MUTANTS BETWEEN BETA-GLU-181 AND BETA-LYS-801 RESIDUES 査読

MY PARK, H OMOTE, M MAEDA, M FUTAI

JOURNAL OF BIOCHEMISTRY 116 ( 5 ) 1139 - 1145 1994年11月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：JAPANESE BIOCHEMICAL SOC

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, Ale, or Lys and beta Arg-182-->Lys, Ala, Glu, or Gin, 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 F-1-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 F-1-ATPases of the beta Gln-181 and beta Ala-181 mutants showed no multisite (or steady state) catalysis and slow unisite catalysis (less than or equal to 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 (less than or equal to 0.1% of the multi- and similar to 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. The position 181 and 182 mutant enzymes had significantly increased K-d (k(-1)/k(+1)) values, reflecting decreased substrate binding. These results suggest that beta Glu-181 and beta Arg-182 are essential for substrate binding, although mutations with conservative substitutions at these positions do not have drastic effects. This study also indicates the importance of the conserved Gly-Glu-Arg (GER) sequence (beta 180-beta 182).

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CATALYSIS AND ENERGY COUPLING OF H+-ATPASE (ATP SYNTHASE) - MOLECULAR BIOLOGICAL APPROACHES 査読

M FUTAI, MY PARK, A IWAMOTO, H OMOTE, M MAEDA

BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS 1187 ( 2 ) 165 - 170 1994年8月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：ELSEVIER SCIENCE BV

The molecular biological approach has provided important information for understanding the FoF1 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.

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THE ALPHA/BETA SUBUNIT INTERACTION IN H+-ATPASE (ATP SYNTHASE) - AN ESCHERICHIA-COLI ALPHA-SUBUNIT MUTATION (ARG-ALPHA-296 -] CYS) RESTORES COUPLING EFFICIENCY TO THE DELETERIOUS BETA-SUBUNIT MUTANT (SER-BETA-174 -] PHE) 査読

H OMOTE, MY PARK, M MAEDA, M FUTAI

JOURNAL OF BIOLOGICAL CHEMISTRY 269 ( 14 ) 10265 - 10269 1994年4月

記述言語：英語掲載種別：研究論文（学術雑誌）出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

The Ser-beta 174 residue of the Escherichia coli H+-ATPase beta subunit has been shown to be near the catalytic site together with Gly-beta 149, Gly-beta 172, Glu-beta 192, and Val-beta 198 (Iwamoto, A., Park, M.-Y., Maeda, M., and Futai, M. (1993) J. Biol. Chem. 268, 3156-3160). In this study, we introduced various residues at position 174 and found that the larger the side chain volume of the residue introduced, the lower the enzyme activity became. The Phe-beta 174 mutant was defective in energy coupling between catalysis and transport, whereas the Leu-beta 174 mutant could couple efficiently, although both mutants had essentially the same ATPase activities (similar to 10% of the wild type). The defective energy coupling of the Phe-beta 174 mutant was suppressed by the second mutation (Arg-alpha 296 --> Cys) in the alpha subunit. The Cys-alpha 296/Phe-beta 174 mutant had essentially the same membrane ATPase activity as the Phe-beta 174 single mutant when assayed under the conditions that stabilize the double mutant enzyme. These results indicate the importance of the alpha/beta interaction, especially that between the regions near Arg-alpha 296 and Ser-beta 174, for energy coupling in the H+-ATPase. The 2 residues (Ser-beta 174 and Arg-alpha 296) may be located nearby at the interface of the two subunits. About 1 mol of N-[C-14]ethylmaleimide could bind to 1 mol of the alpha subunit of Cys-alpha 296/Phe-beta 174 or Cys-alpha 296 mutant ATPase, but could not inhibit the enzyme activity. This is the first intersubunit mutation/suppression approach to ATPase catalysis and its energy coupling.

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Osmoenzyme-H(+)-ATP synthase: catalysis and H+ translocation

Futai, M., Omote, H., Maeda, M.

Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme 39 ( 7 ) 1994年

ESCHERICHIA-COLI F0F1-ATPASE - RESIDUES INVOLVED IN CATALYSIS AND COUPLING 査読

RK NAKAMOTO, K SHIN, A IWAMOTO, H OMOTE, M MAEDA, M FUTAI

ANNALS OF THE NEW YORK ACADEMY OF SCIENCES 671 335 - 344 1992年11月

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