• Search Research Projects
  • Search Researchers
  • How to Use
  1. Back to project page

1993 Fiscal Year Final Research Report Summary

Changes of Active Site Structure with the Binding of Substrate in Aspartate Aminotransferase

Research Project

Project/Area Number 04680167
Research Category

Grant-in-Aid for General Scientific Research (C)

Allocation TypeSingle-year Grants
Research Field 物質生物化学
Research InstitutionKumamoto University

Principal Investigator

HIGAKI Tsuyosi  KUMAMOTO UNIVERSITY, COLLEGE OF MEDICAL SCIENCE, RESEARCHER, 医療技術短期大学部, 助手 (70128304)

Co-Investigator(Kenkyū-buntansha) TANASE Sumio  KUMAMOTO UNIVERSITY, SCHOOL OF MEDICINE, ASSOCIATE PROFESSOR, 医学部, 助教授 (20112401)
Project Period (FY) 1992 – 1993
KeywordsNuclear magnetic resonace / Aminotransferase / Induced fit / Amino acid substitution / Enzyme structure / Pyridoxal / Catalytic function / Aspartate
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.

  • Research Products

    (4 results)

All Other

All Publications (4 results)

  • [Publications] Yano,T.et al.: "The role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase" Biochemistry. 31. 5879-5887 (1992)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Pan,Q.W.et al.: "Functional roles of Val37 and Gly38 in the mobile loop of porcine cytosolic aspartate aminotransferase" J.Biol.Chem.268. 24758-24765 (1993)

    • Description
      「研究成果報告書概要(和文)」より
  • [Publications] Yano, T.: "The role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase : An amino acid residue which enhances the function of the enzyme-bound coenzyme, pyridoxal 5'-phosphate." Biochemistry. 31(25). 5879-5887 (1992)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Publications] Pan, Q.-W.: "Functional roles ov Va137 and Gly38 in the mobile loop of porcine cytosolic aspartate aminotransferase." J.Biol.Chem.268(33). 24758-24765 (1993)

    • Description
      「研究成果報告書概要(欧文)」より

URL: 

Published: 1995-03-27  

Information User Guide FAQ News Terms of Use Attribution of KAKENHI

Powered by NII kakenhi