Changes of Active Site Structure with the Binding of Substrate in Aspartate Aminotransferase
| Project/Area Number |
04680167
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
物質生物化学
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| Research Institution | Kumamoto University |
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Principal Investigator |
HIGAKI Tsuyosi KUMAMOTO UNIVERSITY, COLLEGE OF MEDICAL SCIENCE, RESEARCHER, 医療技術短期大学部, 助手 (70128304)
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| Co-Investigator(Kenkyū-buntansha) |
TANASE Sumio KUMAMOTO UNIVERSITY, SCHOOL OF MEDICINE, ASSOCIATE PROFESSOR, 医学部, 助教授 (20112401)
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| Project Period (FY) |
1992 – 1993
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| Project Status |
Completed (Fiscal Year 1993)
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| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1993: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1992: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Keywords | Nuclear magnetic resonace / Aminotransferase / Induced fit / Amino acid substitution / Enzyme structure / Pyridoxal / Catalytic function / Aspartate |
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| Research Abstract |
Porcine cytosolic aspartate aminotransferase is composed of two identical subunits of 412 residues. Each subunit cinsists of three domains ; an amino-terminal segment (residues 1-14), a small domain (residues 15-49 and 326-412) and a large domain (residues 50-325). X-ray studies indicated that the substrate binding induces a large conformational change, in which the small domain comes closer to the coenzyme binding site and completes the catalytic site. In this "Induced fit" movement, a peptide stretch, residues 15-18 (Val-Leu-Val-Phe) of the amino-terminal segment, shows a large shift toward the active site, and is situated in the close proximity to the side chain of Arg292 to provide an appropriate environment for the substrate binding and catalysis. Thus, the active site cleft of the enzyme is prone to be hydrophobic and discriminate against solvent during catalysis. Phe18 is of most hydrophobic nature among these residues and shows the largest shift in the position upon the "Induced fit" movement. In the present study, to assess the structural and functional dynamics fo the amino-terminal segment, a site-directed mutagenesis is used to replace Phe18 with Tryptophan (F18W), Tyrosine (F18Y), Histidine (F18H) and Alanine (F18A). Replacement of Phe18 by Ala resulted in a large decrease in both catalytic rate and binding affinity for substrates. F18W, F18Y and F18H showed a moderate decrease in kcat and a considerable increase in km values. ^1H-NMR observations of F18H, in which His18 served as a built-in probe, were in accord with the behavior that would be expected from the conformational transition.
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Report
(2 results)
Research Products
(5 results)
