1990 年度実績報告書

細菌が生産するプロテア-ゼインヒビタ-機能構造についての蛋白工学的研究

研究課題

研究課題/領域番号	02305003
研究機関	東京大学
研究代表者	三浦謹一郎東京大学, 工学部, 教授 (30000227)
研究分担者	赤坂一之京都大学, 理学部, 助教授 (50025368) 外村辨一郎京都大学, 農学部, 教授 (20026545) 甲斐荘正恒東京都立大学, 理学部, 助教授 (20137029) 太田隆久東京大学, 農学部, 教授 (30011844) 三井幸雄長岡技術科学大学, 工学部, 教授 (40012637)
キーワード	蛋白工学 / 酵素阻害剤 / プロテア-ゼ・インヒビタ- / 部位指定変異 / バクテリア / 遺伝子工学 / 蛋白質の機能部位 / 蛋白質の構造
研究概要	プロテア-ゼとそのインヒビタ-の系は蛋白質相互作用のモデルとして好適である。まず種々の細菌からプロテア-ゼやインヒビタ-のスクリ-ニングを行い、カビの一種より基質特異性の非常に高いセリンプロテア-ゼを、土壌菌よりレトロウイルスプロテア-ゼやペプシンなどの酸性プロテア-ゼに対する低分子量のインヒビタ-を、海洋細菌よりパパインを阻害するペプチド性のインヒビタ-を分離精製した。この一部については構造決定を行った。また有機合成法によりプロリルエンドペプチダ-ゼのインヒビタ-の構造活性相関の研究を行った。金属プロテア-ゼに対する蛋白性のインヒビタ-SMPIについてはその遺伝子をクロ-ニングし、塩基配列も決定した。さらに、その構造について詳細に研究されている<Streptomyces>___ー Subtilisin Inhibitor(SSI)については、種々の変異体を作製し構造と機能の相関について詳細に解析するという蛋白工学的研究を進めた。まず作用部位周辺のアミノ酸残基を変換し、ズブチリシン、アクアライシンの他、金属プロテア-ゼに対する阻害活性やkineticsを測定した結果、プロテア-ゼの基質特異性との高い相関関係が明らかになった。P1部位がLysである変異SSIについてはカロリメトリ-によりズブチリシン及びトリプシンとの相互作用について熱力学特性を調べた。またこの変異体とズブチリシンの複合体のX線構造解析を行い、野生型との局所的な構造変化の違いが明らかになった。Trp86→Hisの変異体については種々の物理化学的測定により、この変異体の一時阻害がその構造不安定化によることが示された。また安定同位体ラベルとアミノ酸置換体の組合せにより、主鎖のカルボニル炭素のNMRシグナルの帰属を進め、構造変化についても検討した。以上のようにSSIについては変異体の利用によって多角的に構造と機能の関係を追究している。

研究成果
(26件)

すべてその他

すべて文献書誌 (26件)

[文献書誌] K.Cda,Y.Fukuda,S.Murao K.Uchida and M.Kainosho: "A Novel Proteinase Inhibitor,Inhibiting Some PepstatinーInsensitive Carboxyl Proteinases" Agric.Biol.Chem.53. 405-415 (1989)
[文献書誌] D.Tsuru,A.Naotuka,R.Kobayashi,T.Yoshimoto,K.Oda and S.Murao: "Inactivation of <Scytalidium>___ー <lignicolum>___ー Acid Protease B with 1,2ーEpoxyー3ー(4′ーdiazoー2′ーnitrophenoxy)propane" Agric.Biol.Chem.53. 2751-2756 (1989)
[文献書誌] 村尾沢夫,小田耕平: "放線菌由来のメタルプロテア-ゼインヒビタ-" 蛋白質・核酸・酵素. 34. 980-988 (1989)
[文献書誌] S.ーH.Kim,S.Hara,T.Ikenaka: "Reconstitution of new inhibitors by mutual exchange of Ile(64)ーMet(84) peptides of soybean trypsin inhibitors,Ti^a and Ti^b." J.Biochem.
[文献書誌] R.Takano,C.Imada,K.Kamei,S.Hara: "The reactive site of the novel peptide serine proteinase inhibitor,marinostatin" J.Biochem.
[文献書誌] T.Yoshimoto,D.Tsuru,N.Yamamoto,R.Ikezawa,S.Furukawa: "Structure activity relationship of inhibitors specific for prolyl endopeptidase." Agricultural Biological Chemistry. 55. 37-43 (1991)
[文献書誌] K.Akasaka,R.Ishima,and S.Shibata,: "Proton spin relaxation in proteins at high mangetic fields." Physica B. 164,. 163-177 (1990)
[文献書誌] S.Shibata and K.Akasaka,: "Computer simulation program of proton spin relaxation in proteins and its application to Streptomyces subtilisin inhibitor." Magnetic Resonance in Chemistry. 28,. 129-137 (1990)
[文献書誌] R.Ishima,S.Shibata,and K.Akasaka,: "General features of proton longitudinal relaxation in proteins in solution." J.Magn.Reson.,in press.91. (1991)
[文献書誌] S.Murao,K.Ohkuni,T.Shin: "Characterization of thermostable acid proteinase from thermophilic <Bacillus>___ー novosp.MNー32." Agric.Biol.Chem.
[文献書誌] I.Terada,SukーTae Kwon,Y.Miyata,H.Matsuzawa,T.Ohta: "Unique precursor structure of an extracellular protease,aqualysin I.with NH_2ーand COOHーterminal proーsequences and its processing in <Escherichia>___ー <coli>___ー." J.Biol.Chem.265. 6576-6581 (1990)
[文献書誌] 加藤,松永,西村,ベルヒリ,甲斐荘,荒田: "Application of ^<13>C nuclear maguetic resonance spectroscopy to molecular structural auslyses of autibody molecules." J.Biochem.105. 867-869 (1989)
[文献書誌] 小田,福田,村尾,内田,甲斐荘: "A novel proteinase inhibitor,tyrostatin,inhibiting some pepstatinーinsensitime proteinases." Agric.Bilo.Chem.53. 405-415 (1989)
[文献書誌] 内田,三宅,甲斐荘: "Reductive cleavage and regeneration of the disulfide bonds in <Streptomyces>___ー subtilisin inhibitor (SSI) as studied by the carbonyl ^<13>C NMR resonances of cysteinyl residues." J.Biomol.NMR. 1. 000-000 (1991)
[文献書誌] 内田,甲斐荘: "Synthesis of 〔1,1′ー^<13>C_2〕ーLーCystive." J.Labelled Compd.Radiopharmacenticals.
[文献書誌] S.Taguchi,I.Kumagai,K.Miura: "Comparison of secretory expression in Escherichia coli and Streptomyces of streptomyces subtilisin inhibitor (SSI) gene." Biochim.Biophys.Acta. 1049. 278-285 (1990)
[文献書誌] S.Kojima,I.Kumagai,K.Miura: "Effect on inhibitory activity of mutation at reaction site P4 of tne streptomyces subtilisin inhibitor,SSI." Protein Engineering. 3. 527-530 (1990)
[文献書誌] I.Kumagai,S.Takeda,T.Hibino,K.Miura: "Expression of goat αーlactalbumin in Escherichia coli and its refolding to biologically active protein." Protein Engineering. 3. 449-452 (1990)
[文献書誌] S.Kojima,S.Obata,I.Kumagai,K.Miura: "Alteration of the specificity of the streptomyces subtilisin inhibitor by gene engineering." BIO/TECHNOLOGY. 8. 449-452 (1990)
[文献書誌] S.Taguchi,I.Kumagai,J.Nakayama,A.Suzuki,K.Miura: "Efficient extracellular expression of a foreign protein in Streptomyces using secretory protease inhibitor (SSI) gene fusions." BIO/TECHNOLOGY. 7. 1063-1066 (1989)
[文献書誌] S.Taguchi,K.Nishiyama,I.Kumagai,K.Miura: "Analysis of transctiptional control regions in the <Streptomyces>___ー subtilisinーinhibitorーencoding gene." Gene. 84. 279-286 (1989)
[文献書誌] Y.Yoshinaka,I.Katoh and K.Oda: "Retroviral Proteases (ed.L.H.Pearl) Retroviral Protease:Substrate Specificity and Inhibitorsの項執筆" Stockton Press, 9 (1990)
[文献書誌] K.Oda and S.Murao: "Aspartic Proteinases (ed.B.M.Dunn) Pepstatinーinsensitive Carboxyl Proteinasesの項執筆" Plenum Press, 16 (1991)
[文献書誌] 鶴大典: "プロテア-ゼとそのインヒビタ-" メジカルビュ-社, 20 (1991)
[文献書誌] 甲斐荘,内田(共著): "生物物理のフロンティア" 培風館, 291 (1989)
[文献書誌] 甲斐荘,三宅(共著): "新生化学実験講座I,タンパク質III 高次構造" 東京化学同人, 436 (1990)

1990 年度実績報告書

細菌が生産するプロテア-ゼインヒビタ-機能構造についての蛋白工学的研究

研究代表者

三浦謹一郎東京大学, 工学部, 教授 (30000227)

研究成果

[文献書誌] K.Cda,Y.Fukuda,S.Murao K.Uchida and M.Kainosho: "A Novel Proteinase Inhibitor,Inhibiting Some PepstatinーInsensitive Carboxyl Proteinases" Agric.Biol.Chem.53. 405-415 (1989)

[文献書誌] D.Tsuru,A.Naotuka,R.Kobayashi,T.Yoshimoto,K.Oda and S.Murao: "Inactivation of <Scytalidium>_ー <lignicolum>_ー Acid Protease B with 1,2ーEpoxyー3ー(4′ーdiazoー2′ーnitrophenoxy)propane" Agric.Biol.Chem.53. 2751-2756 (1989)

[文献書誌] 村尾沢夫,小田耕平: "放線菌由来のメタルプロテア-ゼインヒビタ-" 蛋白質・核酸・酵素. 34. 980-988 (1989)

[文献書誌] S.ーH.Kim,S.Hara,T.Ikenaka: "Reconstitution of new inhibitors by mutual exchange of Ile(64)ーMet(84) peptides of soybean trypsin inhibitors,Ti^a and Ti^b." J.Biochem.

[文献書誌] R.Takano,C.Imada,K.Kamei,S.Hara: "The reactive site of the novel peptide serine proteinase inhibitor,marinostatin" J.Biochem.

[文献書誌] T.Yoshimoto,D.Tsuru,N.Yamamoto,R.Ikezawa,S.Furukawa: "Structure activity relationship of inhibitors specific for prolyl endopeptidase." Agricultural Biological Chemistry. 55. 37-43 (1991)

[文献書誌] K.Akasaka,R.Ishima,and S.Shibata,: "Proton spin relaxation in proteins at high mangetic fields." Physica B. 164,. 163-177 (1990)

[文献書誌] S.Shibata and K.Akasaka,: "Computer simulation program of proton spin relaxation in proteins and its application to Streptomyces subtilisin inhibitor." Magnetic Resonance in Chemistry. 28,. 129-137 (1990)

[文献書誌] R.Ishima,S.Shibata,and K.Akasaka,: "General features of proton longitudinal relaxation in proteins in solution." J.Magn.Reson.,in press.91. (1991)

[文献書誌] S.Murao,K.Ohkuni,T.Shin: "Characterization of thermostable acid proteinase from thermophilic <Bacillus>___ー novosp.MNー32." Agric.Biol.Chem.

[文献書誌] I.Terada,SukーTae Kwon,Y.Miyata,H.Matsuzawa,T.Ohta: "Unique precursor structure of an extracellular protease,aqualysin I.with NH_2ーand COOHーterminal proーsequences and its processing in <Escherichia>_ー <coli>_ー." J.Biol.Chem.265. 6576-6581 (1990)

[文献書誌] 加藤,松永,西村,ベルヒリ,甲斐荘,荒田: "Application of ^<13>C nuclear maguetic resonance spectroscopy to molecular structural auslyses of autibody molecules." J.Biochem.105. 867-869 (1989)

[文献書誌] 小田,福田,村尾,内田,甲斐荘: "A novel proteinase inhibitor,tyrostatin,inhibiting some pepstatinーinsensitime proteinases." Agric.Bilo.Chem.53. 405-415 (1989)

[文献書誌] 内田,三宅,甲斐荘: "Reductive cleavage and regeneration of the disulfide bonds in <Streptomyces>___ー subtilisin inhibitor (SSI) as studied by the carbonyl ^<13>C NMR resonances of cysteinyl residues." J.Biomol.NMR. 1. 000-000 (1991)

[文献書誌] 内田,甲斐荘: "Synthesis of 〔1,1′ー^<13>C_2〕ーLーCystive." J.Labelled Compd.Radiopharmacenticals.

[文献書誌] S.Taguchi,I.Kumagai,K.Miura: "Comparison of secretory expression in Escherichia coli and Streptomyces of streptomyces subtilisin inhibitor (SSI) gene." Biochim.Biophys.Acta. 1049. 278-285 (1990)

[文献書誌] S.Kojima,I.Kumagai,K.Miura: "Effect on inhibitory activity of mutation at reaction site P4 of tne streptomyces subtilisin inhibitor,SSI." Protein Engineering. 3. 527-530 (1990)

[文献書誌] I.Kumagai,S.Takeda,T.Hibino,K.Miura: "Expression of goat αーlactalbumin in Escherichia coli and its refolding to biologically active protein." Protein Engineering. 3. 449-452 (1990)

[文献書誌] S.Kojima,S.Obata,I.Kumagai,K.Miura: "Alteration of the specificity of the streptomyces subtilisin inhibitor by gene engineering." BIO/TECHNOLOGY. 8. 449-452 (1990)

[文献書誌] S.Taguchi,I.Kumagai,J.Nakayama,A.Suzuki,K.Miura: "Efficient extracellular expression of a foreign protein in Streptomyces using secretory protease inhibitor (SSI) gene fusions." BIO/TECHNOLOGY. 7. 1063-1066 (1989)

[文献書誌] S.Taguchi,K.Nishiyama,I.Kumagai,K.Miura: "Analysis of transctiptional control regions in the <Streptomyces>___ー subtilisinーinhibitorーencoding gene." Gene. 84. 279-286 (1989)

[文献書誌] Y.Yoshinaka,I.Katoh and K.Oda: "Retroviral Proteases (ed.L.H.Pearl) Retroviral Protease:Substrate Specificity and Inhibitorsの項執筆" Stockton Press, 9 (1990)

[文献書誌] K.Oda and S.Murao: "Aspartic Proteinases (ed.B.M.Dunn) Pepstatinーinsensitive Carboxyl Proteinasesの項執筆" Plenum Press, 16 (1991)

[文献書誌] 鶴大典: "プロテア-ゼとそのインヒビタ-" メジカルビュ-社, 20 (1991)

[文献書誌] 甲斐荘,内田(共著): "生物物理のフロンティア" 培風館, 291 (1989)

[文献書誌] 甲斐荘,三宅(共著): "新生化学実験講座I,タンパク質III 高次構造" 東京化学同人, 436 (1990)

1990 年度 実績報告書

細菌が生産するプロテア-ゼインヒビタ-機能構造についての蛋白工学的研究

研究代表者

三浦 謹一郎 東京大学, 工学部, 教授 (30000227)

研究成果

[文献書誌] K.Cda,Y.Fukuda,S.Murao K.Uchida and M.Kainosho: "A Novel Proteinase Inhibitor,Inhibiting Some PepstatinーInsensitive Carboxyl Proteinases" Agric.Biol.Chem.53. 405-415 (1989)

[文献書誌] D.Tsuru,A.Naotuka,R.Kobayashi,T.Yoshimoto,K.Oda and S.Murao: "Inactivation of <Scytalidium>___ー <lignicolum>___ー Acid Protease B with 1,2ーEpoxyー3ー(4′ーdiazoー2′ーnitrophenoxy)propane" Agric.Biol.Chem.53. 2751-2756 (1989)

[文献書誌] 村尾 沢夫,小田 耕平: "放線菌由来のメタルプロテア-ゼインヒビタ-" 蛋白質・核酸・酵素. 34. 980-988 (1989)

[文献書誌] S.ーH.Kim,S.Hara,T.Ikenaka: "Reconstitution of new inhibitors by mutual exchange of Ile(64)ーMet(84) peptides of soybean trypsin inhibitors,Ti^a and Ti^b." J.Biochem.

[文献書誌] R.Takano,C.Imada,K.Kamei,S.Hara: "The reactive site of the novel peptide serine proteinase inhibitor,marinostatin" J.Biochem.

[文献書誌] T.Yoshimoto,D.Tsuru,N.Yamamoto,R.Ikezawa,S.Furukawa: "Structure activity relationship of inhibitors specific for prolyl endopeptidase." Agricultural Biological Chemistry. 55. 37-43 (1991)

[文献書誌] K.Akasaka,R.Ishima,and S.Shibata,: "Proton spin relaxation in proteins at high mangetic fields." Physica B. 164,. 163-177 (1990)

[文献書誌] S.Shibata and K.Akasaka,: "Computer simulation program of proton spin relaxation in proteins and its application to Streptomyces subtilisin inhibitor." Magnetic Resonance in Chemistry. 28,. 129-137 (1990)

[文献書誌] R.Ishima,S.Shibata,and K.Akasaka,: "General features of proton longitudinal relaxation in proteins in solution." J.Magn.Reson.,in press.91. (1991)

[文献書誌] S.Murao,K.Ohkuni,T.Shin: "Characterization of thermostable acid proteinase from thermophilic <Bacillus>___ー novosp.MNー32." Agric.Biol.Chem.

[文献書誌] I.Terada,SukーTae Kwon,Y.Miyata,H.Matsuzawa,T.Ohta: "Unique precursor structure of an extracellular protease,aqualysin I.with NH_2ーand COOHーterminal proーsequences and its processing in <Escherichia>___ー <coli>___ー." J.Biol.Chem.265. 6576-6581 (1990)

[文献書誌] 加藤,松永,西村,ベルヒリ,甲斐荘,荒田: "Application of ^<13>C nuclear maguetic resonance spectroscopy to molecular structural auslyses of autibody molecules." J.Biochem.105. 867-869 (1989)

[文献書誌] 小田,福田,村尾,内田,甲斐荘: "A novel proteinase inhibitor,tyrostatin,inhibiting some pepstatinーinsensitime proteinases." Agric.Bilo.Chem.53. 405-415 (1989)

[文献書誌] 内田,三宅,甲斐荘: "Reductive cleavage and regeneration of the disulfide bonds in <Streptomyces>___ー subtilisin inhibitor (SSI) as studied by the carbonyl ^<13>C NMR resonances of cysteinyl residues." J.Biomol.NMR. 1. 000-000 (1991)

[文献書誌] 内田,甲斐荘: "Synthesis of 〔1,1′ー^<13>C_2〕ーLーCystive." J.Labelled Compd.Radiopharmacenticals.

[文献書誌] S.Taguchi,I.Kumagai,K.Miura: "Comparison of secretory expression in Escherichia coli and Streptomyces of streptomyces subtilisin inhibitor (SSI) gene." Biochim.Biophys.Acta. 1049. 278-285 (1990)

[文献書誌] S.Kojima,I.Kumagai,K.Miura: "Effect on inhibitory activity of mutation at reaction site P4 of tne streptomyces subtilisin inhibitor,SSI." Protein Engineering. 3. 527-530 (1990)

[文献書誌] I.Kumagai,S.Takeda,T.Hibino,K.Miura: "Expression of goat αーlactalbumin in Escherichia coli and its refolding to biologically active protein." Protein Engineering. 3. 449-452 (1990)

[文献書誌] S.Kojima,S.Obata,I.Kumagai,K.Miura: "Alteration of the specificity of the streptomyces subtilisin inhibitor by gene engineering." BIO/TECHNOLOGY. 8. 449-452 (1990)

[文献書誌] S.Taguchi,I.Kumagai,J.Nakayama,A.Suzuki,K.Miura: "Efficient extracellular expression of a foreign protein in Streptomyces using secretory protease inhibitor (SSI) gene fusions." BIO/TECHNOLOGY. 7. 1063-1066 (1989)

[文献書誌] S.Taguchi,K.Nishiyama,I.Kumagai,K.Miura: "Analysis of transctiptional control regions in the <Streptomyces>___ー subtilisinーinhibitorーencoding gene." Gene. 84. 279-286 (1989)

[文献書誌] Y.Yoshinaka,I.Katoh and K.Oda: "Retroviral Proteases (ed.L.H.Pearl) Retroviral Protease:Substrate Specificity and Inhibitorsの項執筆" Stockton Press, 9 (1990)

[文献書誌] K.Oda and S.Murao: "Aspartic Proteinases (ed.B.M.Dunn) Pepstatinーinsensitive Carboxyl Proteinasesの項執筆" Plenum Press, 16 (1991)

[文献書誌] 鶴 大典: "プロテア-ゼとそのインヒビタ-" メジカルビュ-社, 20 (1991)

[文献書誌] 甲斐荘,内田(共著): "生物物理のフロンティア" 培風館, 291 (1989)

[文献書誌] 甲斐荘,三宅(共著): "新生化学実験講座I,タンパク質III 高次構造" 東京化学同人, 436 (1990)

1990 年度実績報告書

三浦謹一郎東京大学, 工学部, 教授 (30000227)

[文献書誌] D.Tsuru,A.Naotuka,R.Kobayashi,T.Yoshimoto,K.Oda and S.Murao: "Inactivation of <Scytalidium>_ー <lignicolum>_ー Acid Protease B with 1,2ーEpoxyー3ー(4′ーdiazoー2′ーnitrophenoxy)propane" Agric.Biol.Chem.53. 2751-2756 (1989)

[文献書誌] 村尾沢夫,小田耕平: "放線菌由来のメタルプロテア-ゼインヒビタ-" 蛋白質・核酸・酵素. 34. 980-988 (1989)

[文献書誌] I.Terada,SukーTae Kwon,Y.Miyata,H.Matsuzawa,T.Ohta: "Unique precursor structure of an extracellular protease,aqualysin I.with NH_2ーand COOHーterminal proーsequences and its processing in <Escherichia>_ー <coli>_ー." J.Biol.Chem.265. 6576-6581 (1990)

[文献書誌] 鶴大典: "プロテア-ゼとそのインヒビタ-" メジカルビュ-社, 20 (1991)