| Project/Area Number |
05453209
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Structural biochemistry
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| Research Institution | The University of Tokyo |
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Principal Investigator |
TAKAHASHI Kenji The University of Tokyo Graduate School of Science Professor, 大学院・理学系研究科, 教授 (70011533)
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| Co-Investigator(Kenkyū-buntansha) |
MURAMATSU Tomonari The University of Tokyo Graduate School of Science Assistant, 大学院・理学系研究科, 助手 (70212256)
INOUE Hideshi The University of Tokyo Graduate School of Science Assistant, 大学院・理学系研究科, 助手 (20184765)
TANOKURA Masaru The University of Tokyo Biotechnology Research Center Professor, 生物生産工学センター, 教授 (60136786)
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| Project Period (FY) |
1993 – 1994
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| Project Status |
Completed (Fiscal Year 1994)
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| Budget Amount *help |
¥7,500,000 (Direct Cost: ¥7,500,000)
Fiscal Year 1994: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1993: ¥6,000,000 (Direct Cost: ¥6,000,000)
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| Keywords | Proctase A / Proctase B / carboxyl proteinase / site-directed mutagenesis / autocatalytic activation / active site asparticacid residue / NMR / X-ray crystallography / カルボキシル プロテアーゼ / 活性中心カルボキシル基 / 前駆体活性化 / 部位指定突然異導入法 / アスパラギン酸プロテアーゼ / 部位指定突然変異導入法 |
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| Research Abstract |
1.By specific chemical modifications, carboxyl groups were shoun to be involved in the active site of Proctase A as in the case of Proctase B.Asp14 and Asp 111 in the heavyc hain of Proctase A were indicated to be the catalytic groups by site-directed mutagenesis studies using an E.coli expression system for pro-Proctase A.
2.A cDNA of prepro-Proctase B was cloned and sequenced, giving the complete amino acid sequence of the preproenzyme. Pro-Proctase B was shown to be autocatalytically activated under acidic conditions. Site-direcited mutagenesis studies on pro-Proctase B revealed that Lys 36 and Arg 32 are important for the shroctural stability of the proform and its activation, respectively.
3.The expression system of pro-Proctase A in E.coli was established, and the proenzyme was shown to be autocatalytically activated under acidic conditions like pro-Proctase B.
4.The profiles of denaturation of Proctase A at different pHs and temperatures were in vestigated using various physico-chemical methods in clading NMR and circular dichroism, which revealed unique denaturation profile of the enzyine.
5.Nearly all proton signals in NMR of the light chain of Proctase A were assigned and the three-dcmensional structure of the chain was determined. Further, a method for preparation of isotopically labeled Proctase A using Aspergillus niger was established, and using these ^<15>N and/or ^<13>C-labeled Protase A,the three-dimensional structure was investigated.
6.A unique substrate specificity of Proctase A was elucidatid using oxidized B chain of insulin ets, which in as quite differant from that of Proctase B.In addition, the interaction of an inhibitor peptide with Proctase A was investigated by NMR.
7.Isomorphous metal (Pt and Hg)derivatives of Proctase A were crystallized and the three-dimensional shructure of the enzyrne was partly elarified by X-ray crystallographic analysis.
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