Subunit Interaction and Function Relationship of Aspartate Aminotransferase.

• Project/Area Number: 03680175
• Research Category: Grant-in-Aid for General Scientific Research (C)
• Allocation Type: Single-year Grants
• Research Field: 代謝生物化学
• Research Institution: KUMAMOTO UNIVERSITY
• Principal Investigator: TANASE Sumio KUMAMOTO UNIVERSITY, SCHOOL OF MEDICINE, ASSISTANT PROFESSOR, 医学部, 助教授 (20112401)
• Co-Investigator(Kenkyū-buntansha): HIGAKI Tsuyosi KUMAMOTO UNIVERSITY COLLEGE OF MEDICAL SCIENCE, RESEARCH ASSISTANT, 医療技術短期大学部, 助手 (70128304)
• Project Period (FY): 1991 – 1992
• Project Status: Completed (Fiscal Year 1992)
• Budget Amount: ¥1,900,000 (Direct Cost: ¥1,900,000); Fiscal Year 1992: ¥800,000 (Direct Cost: ¥800,000); Fiscal Year 1991: ¥1,100,000 (Direct Cost: ¥1,100,000)
• Keywords: Aspartic acid / Aminotransferase / Pyridoxal / Catalytic activity / Enzyme structure / Genetic engineering / Amino acid substitution / Induced fit / アスパラギン酸アミノ基転移酵素 / ピリドキサ-ルりん酸 / サブユニット間相互作用

Research Abstract

Porcine cytosolic aspartate aminotransferase is composed of two identical subunits of 412 residues. Each subunit consists of three domains ; an aminoterminal 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 amino-terminal region may involve in the subunit interaction and His68 is located within a distance of strong hydrogen bonding with the residues of neighboring subunit. In the present study, the functional role of some amino acid residues in the subunit interaction were probed by applying the site-directed in vitro mutation technique for amino acid substitutions.
(1) Deletion of amino-terminal residues revealed that an amino-terminal region is particularly important for the maintenance of structural integrity as well as the correct movement of the small domain that is closely related to substrate binding and catalysis.
(2) Substitution of His68 suggested that the strong counter ionic interaction with residues of neighboring subunit may contribute to intersubunit interaction and to maintenance of a rigid and proper structure of the enzyme.
(3) Limited proteolysis experiment of His68 mutant showed that deletion of aminoterminal region of the mutant enzyme resulted in a little decrease of catalytic activity as compared with the case of wild-type enzyme. Gel filtration study indicated that monomeric structure of truncated His68 mutant enzyme. However, truncated enzyme showed very week stability to heat and pH change.

Research Products

• [Publications] Yano,T: "The role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase:Amino-acid residue which enhances the function of the enzyme-bound coenzyme,pyridoxal 5'-phosphate." Biochemistry. 31. 5879-5887 (1992)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] 棚瀬 純男: "アミノ酸代謝酵素の分子生物学" 日本臨床. 50. 48-56 (1992)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Yano,T: "The role of His143 in the catalytic mechanism of Escherichia coli aspartate aminotransferase." J.Biol.Chem.266. 6079-6085 (1991)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Higaki,T.: "Porcine cytosolic aspartate aminotransferase reconstituted with [4'-13C]pyridoxal phosphate.pH-and ligand-induced changes of the coenzyme observed by 13C NMR spectroscopy." Biochemistry. 30. 2519-2526 (1991)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Fukumoto,Y.: "Structural and functional role of the amino-terminal region of porcine cytosolic aspartate aminotransferase.Catalytic and structural properties of enzyme derivatives truncated on the amino-terminal side" J.Biol.Chem.266. 4187-4193 (1991)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Morino,Y.: "Progress in Clinical Biochemistry(K.Miyata,T.Kanno,E.Ishikawa eds)Molecular biology of aspartate aminotransferase isozymes:Structural comparison of isozymic genes and molecular basis of catalysis." Elsevier Science Publishers,B.V, 7 (1992)
  • Description: 「研究成果報告書概要(和文)」より
• [Publications] Fukumoto, Y., Tanase, S., Nagashima, F., Ueda, S., Ikegami, K., & Morino, Y.: "Structural and functional role of the amino-terminal region of porcine cytosolic aspartate aminotransferase. Catalytic and structural properties of enzyme derivatives truncated on the amino-terminal side." J. Biol. Chem.266(7). 4187-4193 (1991)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Higaki, T., Tanase, S., Nagashima, F., Morino, Y., Scott, A. I., Williams, H. J., & Stolowich, N. J.: "Porcine cytosolic aspartate aminotransferase reconstituted with [4'-13C]pyridoxal phosphate. pH- and ligand-induced changes of the coenzyme observed by 13C NMR spectroscopy." Biochemistry. 30(9). 2519-2526 (1991)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Yano, T., Kuramitsu, S., Tanase, S., Morino, Y., Hiromi, K., & Kagamiyama, H.: "The role of His143 in the catalytic mechanism of Escherichia coli aspartate aminotransferase." J. Biol. Chem.266(10). 6079-6085 (1991)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Yano, T., Kuramitsu, S., Tanase, S., Morino, Y., & Kagamiyama, H.: "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). 5878-5887 (1992)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Tanase, S. and Y. Morino: "Molecular biological aspects of amino acid metabolizing enzymes." Nihon Rinsyo. 50(7). 1488-1496 (1992)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Morino, Y. and Tanase, S.: "Molecular biology of aspartate aminotransferase isozymes : Structural comparison of isozymic genes and molecular basis of catalysis. (K. Miyata, T. Kano, E. Ishikawa eds)" Progress in Clinical Biochemistry Elsevier Science Publishers, B. V. 357-362 (1992)
  • Description: 「研究成果報告書概要(欧文)」より
• [Publications] Yano.T: "The role of Asp222 in the catalytic mechanism of Escherichia coli aspartate aminotransferase:Amino-acid residue which enhances the function of the enzyme-bound coenzyme,pyridoxal 5′-phosphate." Biochemistry. 31. 5879-5887 (1992)
• [Publications] 棚瀬 純男: "アミノ酸代謝酵素の分子生物学" 日本臨床. 50. 48-56 (1992)
• [Publications] Morino,Y.: "Progress in Clinical Biochemistry(K.Miyata,T.Kanno,E.Ishikawa eds)Molecular biology of aspartate aminotransferase isozymes:Structural comparison of isozymic genes and molecular basis of catalysis." Elsevier Science Publishers,B.V, 7 (1992)
• [Publications] Fukumoto,Y.: "Structural and functional role of the aminoーterminal region of porcine cytosolic aspartate aminotransferase.Catalytic and structural properties of enzyme derivatives truncated on the aminoーterminal side." J.Biol.Chem.266. 4187-4193 (1991)
• [Publications] Higaki T.: "Porcine cytosolic aspartate aminotransferase reconstituted with[4'-13C]pyridoxal phosphate.pHーand ligandーinduced changes of the coenzyme observed by 13C NMR spectroscopy." Biochemistry. 30. 2519-2526 (1991)
• [Publications] Yano,T.: "The role of His143 in the catalytic mechanism of Escherichia coli aspartate aminotransferase." J.Biol.Chem.266. 6079-6085 (1991)
• [Publications] Yano,T.: "The role of Asp222 in the catalytic mechamism of Escherichia coli aspartate aminotransferase:An amino acid residue which enhances the function of the enzymeーbound coenzyme,pyridoxal 5'ーphosphate." Biochemistry. 31. (1992)