| Project/Area Number |
02044117
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Institution | KUMAMOTO UNIVERSITY |
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Principal Investigator |
MORINO Yoshimasa KUMAMOTO UNIVERSITY, PRESIDENT, 学長 (30028352)
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| Co-Investigator(Kenkyū-buntansha) |
KIRSCH J.F. UNIVERSITY OF CALIFORNIA, PROFESSOR, 生化学, 教授
ARNONE A. THE UNIVERSITY OF IOWA, PROFESSOR, 生化学, 教授
METZLER D.E. IOWA STATE UNIVERSITY, PROFESSOR, 生化学, 教授
HIROTSU Ken OSAKA CITY UNIVERSITY, FACULTY OF SCIENCE, PROFESSOR, 理学部, 教授 (10047269)
YANO Takato OSAKA MEDICAL COLLEGE, RESEARCH ASSISTANT, 助手 (40239827)
HAYASHI Hideyuki OSAKA MEDICAL COLLEGE, LECTURER, 講師 (00183913)
KURAMITSU Seiki OSAKA UNIVERSITY, FACULTY OF SCIENCE, PROFESSOR, 理学部, 教授 (60153368)
TANASE Sumio KUMAMOTO UNIV., SCHOOL OF MEDICINE, ASSISTANT PROFESSOR, 医学部, 助教授 (20112401)
KAGAMIYAMA Hiroyuki OSAKA MEDICAL COLLEGE, PROFESSOR, 教授 (80028555)
KIRSCH J.F. カルフォルニア大学, 生化学, 教授
樋口 泰一 大阪市立大学, 理学部, 教授 (40046868)
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| Project Period (FY) |
1990 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥9,000,000 (Direct Cost: ¥9,000,000)
Fiscal Year 1992: ¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 1991: ¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1990: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Keywords | Aminotransferse / Pyridoxal / Genetic engineering / Amino acid substitution / Catalytic activity / Substrate specificity / Structure and function relationship / X-ray crystallography / アスパラギン酸 / ピリドキサ-ル / 酵素構造 / ビタミンB6 |
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| Research Abstract |
Aspartate aminotransferase (AspAT) is one of the most typical pyridoxal phosphate-dependent enzymes. The aim of the present project is to elucidate the structure-function relationships of pig cytosolic Escherichia coli AspATs. The joint research was performed by three research groups in Japan ant three groups in the United States. They include investigators with expertise in x-ray cystallography, site-directed mutagensis, NMR and other spectroscopic techniques. Both enzymes from different sources are very similar in the three dimensional structure ; ie., each subunit of these dimeric proteins consists of a small domain (residues 12-49 and 326-412), and a large domain (residues 50-325) that provides the binding site for the coenzyme. A comparison of the x-ray structure between the free enzyme and the complex with 2-methylaspartate suggested that substrate induces a movement of the small domain toward the bound coenzyme to close the entrance to the active site cleft. Among small domain residues, mutation at Gly38 caused a striking decrease in catalytic activity. X-ray and solution studies showed that Gly38 mutant enzymes bind 2-methylaspartate at active site, but the binding does not induce the closure of the small domain. Gly38 is probably required for proper function of the enzyme because it permits a high level of flexibility for the 36-39 peptide, which in turn allows the essential domain closure. Further x-ray studies have led to a new finding that the peptide stretch from residues 225 to 229 changes its positions upon binding 2-methylaspartate. Another promising issue is inquiry into the catalytic significance of the histidine cluster (His 143, and 189) at the bottom of the coenzyme. Single, double and triple mutations at these positions are currently being prepared. Thus the current results will open a new prospect in pursuing the significance of substrate-induced conformational change, one most intriguing issue in enzymology.
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