| Project/Area Number |
10680585
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | KUMAMOTO UNIVERSITY |
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Principal Investigator |
TANASE Sumio KUMAMOTO UNIVERSITY, SCHOOL OF MEDICINE, ASSOCIATE PROFESSOR, 医学部, 助教授 (20112401)
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| Co-Investigator(Kenkyū-buntansha) |
HIGAKI Tsuyosi KUMAMOTO UNIVERSITY COLLEGE OF MEDICAL SCIENCE, ASSOCIATE PROFESSOR, 医療技術短期大学部, 助教授 (70128304)
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| Project Period (FY) |
1998 – 2000
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| Project Status |
Completed (Fiscal Year 2000)
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| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2000: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1999: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1998: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | Alanine / Aminotransferase / Substrate specificity / Protein engineering / Mutant enzyme / Coenzyme / Vitamin B6 / PCR / アラニン酸 |
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| Research Abstract |
In an animotransferase enzyme group, studies of the aspartate aminotransferase (AspAT) enzyme progress most. Crystal structure and site-directed mutagenesis analysis of the enzyme reveled that correlation with a catalytic mechanism and enzyme structure. However, elucidation of relation of enzyme structure and substrate specificity of other mammalian aminotransferase does not advance due to the delay of three-dimensional structural analysis and insufficient supply of enzyme protein to the analysis. To clarify above limitation to the investigation of Alanine aminotransferase (AlaAT), we have tried to construct overproduction system of human AlaAT in E.coli and Yeast. Furthermore we attempted to make mutant enzyme proteins and identify the structural requirement to the rigid specificity and recognition of substrates in AlaAT.
As a first step toward this ultimate goal, we constructed expression vectors having full length of cDNA coded human AlaAT using PCR technique, and made recombinants of E.coli and Yeast cells. But quantity and catalytic properties of enzyme protein expressed in these cells were not sufficient to the crystallographic study and functional analysis of the enzyme. We changed cDNA sequence considering codon usage, and once again, carried out a sequence analysis and correction of the sequence. We incorporated it to expression vectors having other promoter region used above, and reinvestigated culture condition of recombinant cells. Some enzymatic activity and reaction products to the anti-AlaAT antibody were observed. We proposed substrate recognition site by homology search of the sequence.
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