2003 Fiscal Year Annual Research Report

マトリックスメタロプロテイナーゼ-7の構造と機能の分子論的解析および阻害剤の開発

Research Project

Project/Area Number	14658203
Research Institution	Kyoto University
Principal Investigator	井上國世京都大学, 農学研究科, 教授 (10223249)
Co-Investigator(Kenkyū-buntansha)	滝田禎亮京都大学, 農学研究科, 助手 (70263126) 榊利之京都大学, 農学研究科, 助教授 (70293909)
Keywords	メタロプロテイナーゼ / 金属酵素 / 細胞外マトリックス / 酵素反応機構 / 癌の転移と浸潤 / チロシン / プロテアーゼ / 亜鉛酵素
Research Abstract	ヒト・マトリックスメタロプロテイナーゼ-7(MMP7)は高い蛋白質分解活性をもち、癌転移に関与することが知られている。本酵素の阻害物質は有効な癌治療薬になる可能がある。pH5-9の広いpH域に特徴的な最適pHを有している。本酵素の反応機構は不明である。我々が行なってきたこれまでの研究から、酸性側の活性解離基としてGlu198またはZnルイス酸、塩基性側の活性解離基としてTyr216やTyr219などの可能性が考えられた。今回、塩基性側の活性解離基の同定と役割を解明することを目的とした。部位特異的変異導入により、活性部位に存在する3個のTyr残基(Tyr193,Tyr216,Tyr219)をそれぞれPheに変換した。野生型酵素では、酸性側pKa(pK_1)は4.4、塩基性側pKa(pK_2)は9.8である。Tyr193PheとTyr216Pheは、野生型とほぼ同じpK_1とpK_2値を示した。一方、Tyr219Pheでは、それぞれ5.7と9.4となり、pK_1が1.3ユニットも塩基性側にシフトした。いずれの変異酵素も最適pHでの酵素活性はほぼ同程度であり、以上の変異導入は固有の酵素活性には影響を及ぼさないことが示された。上で示されたpKaのシフトは、Tyr219が塩基性側の活性解離基ではないこと、またその水酸基が塩基性側解離基に直接的な影響を及ぼさないこと、ただし、Tyr219の水酸基により酸性側解離基はプロトン解離が促進されることが示された。塩基性側解離基の同定は未解明のまま残されたが、反応機構の解析において特異で貴重な知見が得られた。

Research Products
(21 results)

All Other

All Publications (21 results)

[Publications] Y.Matsumiya, K.Inouye 他3名: "Analysis of autodegradation sites of thermolysin and enhancement of its thermostability by modifying Leu155 at an autodegradation site"Journal of Biochemistry. 135/4(印刷中). (2004)
[Publications] H.Oneda, S.Lee, K.Inouye: "Inhibitory effect of 0.19 α-amylase inhibitor from wheat kernel on the activity of porcine pancreas α-amylase inhibitor and its thermal stability"Journal of Biochemistry. 135/3. 421-427 (2004)
[Publications] S.-B.Lee, K.Inouye 他4名: "Antioxidative effects of glycosyl-ascorbic acids synthesized by maltogenic amylase to reduce lipid oxidation and volatiles production in cooked chicken meat."Bioscience, Biotechnology, and Biochemistry. 68/1. 36-43 (2004)
[Publications] M.Kamao, S.Tatematsu, K.Inouye 他7名: "C-3 epimerization of vitamin D3 metabolites and further metabolism of C-3 epimers"Journal of Biological Chemistry. (印刷中). (2004)
[Publications] K.Yamamoto, H.Matsuno, K.Inouye 他4名: "Homology modeling of human 25-hydroxyvitamin D3 1α-hydroxylase(CYP27B1)based on the crystal structure of rabbit CYP2C5."Journal of Steroid Biochemistry. (印刷中). (2004)
[Publications] N.Sawada, T.Kusudo, K.Inouye 他7名: "Novel metabolism of 1α,25-dihydroxyvitamin D3 with C24-C25 bond cleavage catalyzed by human CYP24A1"Journal of Biological Chemistry. (印刷中). (2004)
[Publications] T.Kusudo, T.Sakaki, K.Inouye 他5名: "Metabolism of 20-epimer of 1α, 25-dihydroxyvitamin D3 by CYP24: Species-based difference between humans and rats."Biochem.Biophys.Res.Commun.. 309/4. 885-892 (2003)
[Publications] R.Shinkyo, T.Sakaki, K.Inouye 他2名: "Generation of 2,3,7, 8-TCDD-metabolizing enzyme by modifying rat CYP1A1 through site-directed mutagenesis."Biochem.Biophys.Res.Commun. 308/3. 511-517 (2003)
[Publications] T.Sakaki, N.Sawada, K.Inouye 他7名: "Metabolism of 26,26,26,27,27,27-F6-1α,25-dihydroxyvitamin D3 by CYP24:Species-based difference between humans and rats."Biochemical Pharmacology. 65/. 1957-1965 (2003)
[Publications] H.Oneda, K.Inouye: "Effect of nitreation on the activity of bovine erythrocyte Cu, Zn-superoxide dismutase (BESOD) and kinetic analysis of its dimerization-dissociation reaction as examined by subunit exchange between the native, and nitrated BESODs."Journal of Biochemistry. 134/5. 683-690 (2003)
[Publications] T.Kusudo, T.Sakaki, K.Inouye: "Purification and characterization of purple acid phosphatase PAP1 from dry powder of sweet potato."Bioscience, Biotechnology, and Biochemistry. 67/7. 1609-1611 (2003)
[Publications] K.Nakamura, T.Suzuki, K.Inouye 他3名: "Characterization of p-aminobenzamidine Toyopearl and its use for high-performance affinity chromatography of trypsin-like proteases."Journal of Chromatography A. 1009/. 133-139 (2003)
[Publications] T.Takita, M.Nakagoshi, K.Inouye 他1名: "Lysyl-tRNA synthetase from Bacillus stearothermophilus : The Trp314 residue is shielded in a non-polar environment and is responsible for the fluorescence changes observed in the amino acid activation reaction"Journal of Molecular Biology. 325/. 677-695 (2003)
[Publications] N.Ikezawa, F.Sato, K.Inouye 他4名: "Molecular cloning and characterization of CYP719, a methylenedioxy bridge forming enzyme belonging to a novel P450 family, from cultured Copis japonica cells."Journal of Biological Chemistry. 278/40. 38557-38565 (2003)
[Publications] H.Oneda, M.Shiihara, K.Inouye: "Inhibitory effects of green tea catechins on the activity of human matrix metalloproteinase-7 (matrilysin)."Journal of Biochemistry. 133/5. 571-576 (2003)
[Publications] A.Tanaka, Y.Muta, K.Inouye 他4名: "Differential scanning calorimetry of the effects of Ca^<2+> on the thermal unfolding of Pseudomonas cepacia lipase."Bioscience, Biotechnology, and Biochemistry. 67/1. 207-210 (2003)
[Publications] R.Shinkyo, T.Sakaki, K.Inouye 他1名: "Metabolic pathways of dioxin by CYP1A1. Species difference between rat and human CYP1A subfamily in the metabolism of dioxins."Archives of Biochemistry and Biophysics. 409/. 180-187 (2003)
[Publications] 小根田洋史, 井上國世: "活性のpH依存性がベル型から外れたユニークな酵素の例"化学と生物. 41/4. 250-251 (2003)
[Publications] 井上國世, 小根田洋史: "カテキンによるヒトのガン転移酵素MMP-7の阻害"Foods, Food Ingredients J.Jpn.. 208/12. 975-979 (2003)
[Publications] 永井宏平, 井上國世: "タンパク質性プラスチックの最近の動向"化学と生物. 41/11. 728-730 (2003)
[Publications] K.Inouye: "Handbook of Food Enzymology (J.R.Whitaker et al., Eds.)"Mercel Dekker. 1108 (2003)

2003 Fiscal Year Annual Research Report

マトリックスメタロプロテイナーゼ-7の構造と機能の分子論的解析および阻害剤の開発

Principal Investigator

井上 國世 京都大学, 農学研究科, 教授 (10223249)

Research Products

[Publications] Y.Matsumiya, K.Inouye 他3名: "Analysis of autodegradation sites of thermolysin and enhancement of its thermostability by modifying Leu155 at an autodegradation site"Journal of Biochemistry. 135/4(印刷中). (2004)

[Publications] H.Oneda, S.Lee, K.Inouye: "Inhibitory effect of 0.19 α-amylase inhibitor from wheat kernel on the activity of porcine pancreas α-amylase inhibitor and its thermal stability"Journal of Biochemistry. 135/3. 421-427 (2004)

[Publications] S.-B.Lee, K.Inouye 他4名: "Antioxidative effects of glycosyl-ascorbic acids synthesized by maltogenic amylase to reduce lipid oxidation and volatiles production in cooked chicken meat."Bioscience, Biotechnology, and Biochemistry. 68/1. 36-43 (2004)

[Publications] M.Kamao, S.Tatematsu, K.Inouye 他7名: "C-3 epimerization of vitamin D3 metabolites and further metabolism of C-3 epimers"Journal of Biological Chemistry. (印刷中). (2004)

[Publications] K.Yamamoto, H.Matsuno, K.Inouye 他4名: "Homology modeling of human 25-hydroxyvitamin D3 1α-hydroxylase(CYP27B1)based on the crystal structure of rabbit CYP2C5."Journal of Steroid Biochemistry. (印刷中). (2004)

[Publications] N.Sawada, T.Kusudo, K.Inouye 他7名: "Novel metabolism of 1α,25-dihydroxyvitamin D3 with C24-C25 bond cleavage catalyzed by human CYP24A1"Journal of Biological Chemistry. (印刷中). (2004)

[Publications] T.Kusudo, T.Sakaki, K.Inouye 他5名: "Metabolism of 20-epimer of 1α, 25-dihydroxyvitamin D3 by CYP24: Species-based difference between humans and rats."Biochem.Biophys.Res.Commun.. 309/4. 885-892 (2003)

[Publications] R.Shinkyo, T.Sakaki, K.Inouye 他2名: "Generation of 2,3,7, 8-TCDD-metabolizing enzyme by modifying rat CYP1A1 through site-directed mutagenesis."Biochem.Biophys.Res.Commun. 308/3. 511-517 (2003)

[Publications] T.Sakaki, N.Sawada, K.Inouye 他7名: "Metabolism of 26,26,26,27,27,27-F6-1α,25-dihydroxyvitamin D3 by CYP24:Species-based difference between humans and rats."Biochemical Pharmacology. 65/. 1957-1965 (2003)

[Publications] T.Kusudo, T.Sakaki, K.Inouye: "Purification and characterization of purple acid phosphatase PAP1 from dry powder of sweet potato."Bioscience, Biotechnology, and Biochemistry. 67/7. 1609-1611 (2003)

[Publications] K.Nakamura, T.Suzuki, K.Inouye 他3名: "Characterization of p-aminobenzamidine Toyopearl and its use for high-performance affinity chromatography of trypsin-like proteases."Journal of Chromatography A. 1009/. 133-139 (2003)

[Publications] N.Ikezawa, F.Sato, K.Inouye 他4名: "Molecular cloning and characterization of CYP719, a methylenedioxy bridge forming enzyme belonging to a novel P450 family, from cultured Copis japonica cells."Journal of Biological Chemistry. 278/40. 38557-38565 (2003)

[Publications] H.Oneda, M.Shiihara, K.Inouye: "Inhibitory effects of green tea catechins on the activity of human matrix metalloproteinase-7 (matrilysin)."Journal of Biochemistry. 133/5. 571-576 (2003)

[Publications] A.Tanaka, Y.Muta, K.Inouye 他4名: "Differential scanning calorimetry of the effects of Ca^<2+> on the thermal unfolding of Pseudomonas cepacia lipase."Bioscience, Biotechnology, and Biochemistry. 67/1. 207-210 (2003)

[Publications] R.Shinkyo, T.Sakaki, K.Inouye 他1名: "Metabolic pathways of dioxin by CYP1A1. Species difference between rat and human CYP1A subfamily in the metabolism of dioxins."Archives of Biochemistry and Biophysics. 409/. 180-187 (2003)

[Publications] 小根田洋史, 井上國世: "活性のpH依存性がベル型から外れたユニークな酵素の例"化学と生物. 41/4. 250-251 (2003)

[Publications] 井上國世, 小根田洋史: "カテキンによるヒトのガン転移酵素MMP-7の阻害"Foods, Food Ingredients J.Jpn.. 208/12. 975-979 (2003)

[Publications] 永井宏平, 井上國世: "タンパク質性プラスチックの最近の動向"化学と生物. 41/11. 728-730 (2003)

[Publications] K.Inouye: "Handbook of Food Enzymology (J.R.Whitaker et al., Eds.)"Mercel Dekker. 1108 (2003)

井上國世京都大学, 農学研究科, 教授 (10223249)