1996 Fiscal Year Annual Research Report

NMRとX線によるクロコウジカビ酸性プロテアーゼAの折れたたみ機構の解析

Research Project

Project/Area Number	08272203
Research Institution	The University of Tokyo
Principal Investigator	田之倉優東京大学, 生物生産工学研究センター, 教授 (60136786)
Keywords	酸性プロテアーゼA / NMR / X線非結晶構造解析
Research Abstract	クロコウジカビAspergillus niger var.macrosporusが分泌する酸性プロテアーゼAは基質特異性、阻害剤に対する反応性、一次構造、分子量等の性状が、従来のペプシン型酵素とは著しく異なる新規の酸性プロテアーゼである。本研究では、プロテアーゼAの立体構造、変性中間状態ならびに中性変性過程の解析を行ない、プロテアーゼAの折れたたみ機構を明らかにすることを目指してX線結晶構造解析とX線小角散乱測定を行った。本酵素が活性を示すpH1.5〜3で得られた結晶についてX線回折を測定したところ、1.5Åの分解能の反射が得られた。重原子誘導体の探索を行った結果、Hgおよび2種類のPtの同型置換体が得られた。電子密度図から、この分子はクロワッサン型をしており、αヘリックスを含まずβシートのみからなることが分かった。アスパラギン酸プロテアーゼも主としてβシートからなるクロワッサン型をしており、プロテアーゼAは分子量がアスパラギン酸プロテアーゼに比べて約半分であるにもかかわらず、分子の外形はアスパラギン酸プロテアーゼと大雑把には相似形といえる。ただ、βシート構造の分子全体に対する配向が両者で明らかに異なっており、プロテアーゼAの立体構造法は新規の3次元構造だと考えられる。また、酸性から弱アルカリ性の種々のpHでX線小角散乱の測定を行った。その結果、GuinierプロットからpHが高くなるに従って慣性半径Rgが増大することが示された。またKratkyプロットから、(1)pH4.5ではfolded structure、pH6.75や7.0ではunfolded structureであること、(2)pH6.2では完全なfolded stateでもunfolded stateでもない中間的な状態であること、が分かった。pH4.5からpH8.0への遷移速度をX線小角散乱測定したところ、時定数0.5秒の遷移が見られた。

Research Products
(18 results)

All Other

All Publications (18 results)

[Publications] Ishikawa,K.,Ota,M.,Ariyoshi,Y.,Sasaki.H.,Tanokura,M.,Ming,D.,Caldwell,J.and Abildgaad,F.: "Crystallization and preliminary X-ray analysis of brazzein,a new sweet protein." Acta Crystallographica. D52. 577-578 (1996)
[Publications] Hatano,K.,Tanokura,M.and Takahashi,K.: "Tertiary Structure Comparison of Bromelain Inhibitor VI from Pineapple Stem with Bovine Pancreatic Trypsin Inhibitor." Proceedings of Japan Academy. 72B. 104-107 (1996)
[Publications] Hatano,K.,Kojima,M.,Tanokura,M.and Takahashi,K.: "Solution structure of bromelain inhibitor VI from pineapple stem:Structural similarity with Bowman-Birk trypsin/chymotrypsin inhibitor from soybean" Biochemistry. 35. 5379-5384. (1996)
[Publications] Kukimoto,M.,Nishiyama,M.,Tanokura,M.,Adman,E.T.and Horinouchi,S.: "Studies on protein-protein interaction between copper-containing nitrite reductase and pseudoazurin from Alcaligenes faecalis S-6." Journal of Biolgical Chemistry. 271. 13680-13683 (1996)
[Publications] Ishikawa,K.,Suzuki,E.,Tanokura,M.and Takahashi,K.: "Crystal structure of ribonuclease T1 carboxymethylated at Glu58 in complex with 2'GMP." Biochemistry. 35. 8329-8334. (1996)
[Publications] Koike,H.,Zenno,S.,Saigo,K.and Tanokura,M.: "Crystallization and preliminary crystallographic analysis of the major NAD(P)H:FMN oxidoreductase of Vibrio fischeri ATCC 7744" Journal of Structural Biology. 117. 70-72. (1996)
[Publications] Zenno,S.,Koike,H.,Kumar,A.N.,Jayaraman,R.,Tanokura,M.and Saigo,K.: "Biochemical characterization of NfsA,the Escherichia coli major nitroreductase cxhibiting a high amino acid sequence homology to Frp,a Vibrio harveyi flavin oxidoreductase." Journal of Bacteriology. 178. 4508-4514. (1996)
[Publications] Zenno,S.,Koike,H.,Tanokura,M.and Saigo,K.: "Conversion of NfsB.a minor Escherichia coli nitroreductase,to a flavin reductase similar in biochemical properties to Frase I,the major flavin reductase in Vibrio fischeri,by a single amino acid substitution." Journal of Bacteriology. 178. 4731-4733. (1996)
[Publications] Nishivama,M.,Kinoshita,M.,Kudo,H.,Horinouchi,S.and Tanokura,M.: "Enhancement of the turnover number of thermostable malate dehydrogenase by deleting hydrogen bonds around the catalytic site." Biochemical and Biophysical Research Communications. 225. 844-848. (1996)
[Publications] Kukimoto,M.,Nishiyama,M.,Tanokura,M.,Murphy,M.E.P.,Adman,E.T.and Horinouchi,S.: "Site-dorected mutagenesis of azurin from Pseudomonas aeruginosa enhances the formation of an electron-transfer complex with a copper-containing nitrite reductase from Alcaligenes faecalis S-6." FEBS Letters. 394. 87-90. (1996)
[Publications] Katsumata,T.,Noguchi,S.,Yonezawa,N.,Tanokura,M.and Nakano,M.: "Structural characterization of the N-linked carbohydrate chains of the zona pellucida glycoproteins from bovine ovarian and fertilized eggs." European Journal of Biochemistry. 240. 448-453. (1996)
[Publications] Zenno,S.,Koike,H.,Tanokura,M.and Saigo,K.: "Gene cloning,purification and characterization of NfsB,a minor oxygen-insensitive nitroreductase from Escherichia coli,similar in biochemical properties to Frase I^- the major flavin reductase in Vibrio fischeri." Journal of Biochemistry. 120. 736-744. (1996)
[Publications] Zenno,S.,Koike,H.,Kumar,A.N.,Jayaraman,R.,Tanokura,M.and Saigo,K.: "Biochemical characterization of NfsA,the Escherichia coli major nitroreductase exhibiting a high amino acid sequence homology to Frp,a Vibrio harveyi flavin oxidoreductase." Journal of Bacteriology. 178. 4508-4514. (1996)
[Publications] Zenno,S.,Koike,H.,Tanokura,M.and Saigo,K.: "Conversion of NfsB.a minor Escherichia coli nitroreductase,to a flavin reductase similar in biochemical properties to Frase I,the major flavin reductase in Vibrio fischeri,by a single amino acid substitution." Journal of Bacteriology. 178. 4731-4733. (1996)
[Publications] Nishivama,M.,Kinoshita,M.,Kudo,H.,Horinouchi,S.and Tanokura,M.: "Enhancement of the turnover number of thermostable malate dehydrogenase by deleting hydrogen bonds around the catalytic site." Biochemical and Biophysical Research Communications. 225. 844-848. (1996)
[Publications] Kukimoto,M.,Nishiyama,M.,Tanokura,M.,Murphy,M.E.P.,Adman,E.T.and Horinouchi,S.: "Site-dorected mutagenesis of azurin from Pseudomonas aeruginosa enhances the formation of an electron-transfer complex with a copper-containing nitrite reductase from Alcaligenes faecalis S-6." FEBS Letters. 394. 87-90. (1996)
[Publications] Katsumata,T.,Noguchi,S.,Yonezawa,N.,Tanokura,M.and Nakano,M.: "Structural characterization of the N-linked carbohydrate chains of the zona pellucida glycoproteins from bovine ovarian and fertilized eggs." European Journal of Biochemistry. 240. 448-453. (1996)
[Publications] Zenno,S.,Koike,H.,Tanokura,M.and Saigo,K.: "Gene cloning,purification and characterization of NfsB,a minor oxygen-insensitive nitroreductase from Escherichia coli,similar in biochemical properties to Frase I^- the major flavin reductase in Vibrio fischeri." Journal of Biochemistry. 120. 736-744 (1996)

1996 Fiscal Year Annual Research Report

NMRとX線によるクロコウジカビ酸性プロテアーゼAの折れたたみ機構の解析

Principal Investigator

田之倉 優 東京大学, 生物生産工学研究センター, 教授 (60136786)

Research Products

[Publications] Ishikawa,K.,Ota,M.,Ariyoshi,Y.,Sasaki.H.,Tanokura,M.,Ming,D.,Caldwell,J.and Abildgaad,F.: "Crystallization and preliminary X-ray analysis of brazzein,a new sweet protein." Acta Crystallographica. D52. 577-578 (1996)

[Publications] Hatano,K.,Tanokura,M.and Takahashi,K.: "Tertiary Structure Comparison of Bromelain Inhibitor VI from Pineapple Stem with Bovine Pancreatic Trypsin Inhibitor." Proceedings of Japan Academy. 72B. 104-107 (1996)

[Publications] Hatano,K.,Kojima,M.,Tanokura,M.and Takahashi,K.: "Solution structure of bromelain inhibitor VI from pineapple stem:Structural similarity with Bowman-Birk trypsin/chymotrypsin inhibitor from soybean" Biochemistry. 35. 5379-5384. (1996)

[Publications] Kukimoto,M.,Nishiyama,M.,Tanokura,M.,Adman,E.T.and Horinouchi,S.: "Studies on protein-protein interaction between copper-containing nitrite reductase and pseudoazurin from Alcaligenes faecalis S-6." Journal of Biolgical Chemistry. 271. 13680-13683 (1996)

[Publications] Ishikawa,K.,Suzuki,E.,Tanokura,M.and Takahashi,K.: "Crystal structure of ribonuclease T1 carboxymethylated at Glu58 in complex with 2'GMP." Biochemistry. 35. 8329-8334. (1996)

[Publications] Koike,H.,Zenno,S.,Saigo,K.and Tanokura,M.: "Crystallization and preliminary crystallographic analysis of the major NAD(P)H:FMN oxidoreductase of Vibrio fischeri ATCC 7744" Journal of Structural Biology. 117. 70-72. (1996)

[Publications] Nishivama,M.,Kinoshita,M.,Kudo,H.,Horinouchi,S.and Tanokura,M.: "Enhancement of the turnover number of thermostable malate dehydrogenase by deleting hydrogen bonds around the catalytic site." Biochemical and Biophysical Research Communications. 225. 844-848. (1996)

[Publications] Katsumata,T.,Noguchi,S.,Yonezawa,N.,Tanokura,M.and Nakano,M.: "Structural characterization of the N-linked carbohydrate chains of the zona pellucida glycoproteins from bovine ovarian and fertilized eggs." European Journal of Biochemistry. 240. 448-453. (1996)

[Publications] Nishivama,M.,Kinoshita,M.,Kudo,H.,Horinouchi,S.and Tanokura,M.: "Enhancement of the turnover number of thermostable malate dehydrogenase by deleting hydrogen bonds around the catalytic site." Biochemical and Biophysical Research Communications. 225. 844-848. (1996)

[Publications] Katsumata,T.,Noguchi,S.,Yonezawa,N.,Tanokura,M.and Nakano,M.: "Structural characterization of the N-linked carbohydrate chains of the zona pellucida glycoproteins from bovine ovarian and fertilized eggs." European Journal of Biochemistry. 240. 448-453. (1996)

田之倉優東京大学, 生物生産工学研究センター, 教授 (60136786)