| Project/Area Number |
02454148
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
General medical chemistry
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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) |
TANASE Sumio Kumamoto Univ. Med. School, Lecturer, 医学部, 講師 (20112401)
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| Project Period (FY) |
1990 – 1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥6,900,000 (Direct Cost: ¥6,900,000)
Fiscal Year 1991: ¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1990: ¥4,600,000 (Direct Cost: ¥4,600,000)
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| Keywords | Aspartic acid / Aminotransferase / Pyridoxal / Catalytic activity / Enzyme structure / Genetic engineering / Amino acid substitution / Induced fit / アミノ酸転移酵素 |
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| Research Abstract |
Porcine cytosolic aspartate aminotransferase is composed of two identical subunits of 412 residues. Each subunit consists of three domains ; an amino-terminal segment (residues 1-14), a small domain (residues 15-47 and 326-412) and a large domain (residues 48-325). A conspicuous structural feature of this enzyme is a dynamic interdomain "induced fit" movement during catalysis. In the present study, the functional role of some key amino acid residues within th small domain was probed by applying the site-directed in vitro mutation technique for amino acid substitutions.
(1) Replacement of Vall5 by Asp or Asn, and of Leu16 by Pro led to a large decrease in catalytic activity with concomitant increase in heat stability as well as in resistance to limited proteolysis by protease 401 which cleaves specifically at Leu20 of wild type enzyme. All these functional and structural consequences would result from a decrease in structural flexibility (or plasticity) of the floppy amino-terminal part of the small domain.
(2) Replacement of Phe18 by His, Tyr, or Trp resulted in a small decrease in catalytic activity. Each of residues thus incorporated could be utilized as a spectroscopic reporter group to assess the molecular dynamics. In fact, the ^1H-NMR resonance lines for His18 in the mutant Phe18His showed a dynamic change upon complex formation with a substrate analog, 2-methylaspartate.
(3) Val37 and Gly38 located to a mobile loop of the small domain were replaced by Ala or Ser. In particular, replacement of Gly38 by Ala or Ser resulted in a striking decrease in catalytic activity without affecting the stability of the enzyme. The crystallographic analysis on the mutant enzyme Gly38Ala and Gly38Ser, as performed in collaboration with Dr. Arthur Arnone in the University of Iowa, revealed that the structural change was limited within the side chain of a replacing residue at position 38.
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