| Budget Amount *help |
¥6,500,000 (Direct Cost: ¥6,500,000)
Fiscal Year 1990: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1989: ¥5,100,000 (Direct Cost: ¥5,100,000)
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| Research Abstract |
Aromatic unnatural amino acids were incorporated into phospholipase A_2, and the effect of Photo-illumination on the enzymatic activity was studied. Trp^3 in phospholipase A^2 is known to constitute the interfacial recognition site, which distinguishes phospholipids in the membrane assembly from molecularly dispersed solution.
Mutant enzymes of phospholipase A_2 were prepared by semisynthesis, in which Trp^3 was replaced with naphthylalanine, anthrylalanine, and Phenylazophenylalanine. The outline of the preparation is as follows : i) Amidination selectively on ^<epsilon>-amino groups of Lys residues op phospholipase A_2, ii) elimination of successive three amino acids from the N-ter-minal by Edman degradation (DES3), iii) preparation of BocーAla-Leu-Xaa-OH (Xaa represents aromatic unnatural amino acids) by the conventional liquid phase synthesis, iv) coupling of the tripeptide with DES3, and v) remove of Boc group, and purification of mutant enzymes. Enzymatic activity was analyzed by u
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sing liposome containing [^<14>C]labeled dipalmitoylphosphatidylcholine as substrate. The mutant enzymes replaced by naphthylalanine or anthrylalanine showed a very little activity compared with the native phospholipase A_2. photo-illumination furthermore diminished the enzymatic activity. Presumably, the unnatural amino acid incorporated in the phospholipase A_2 might form an exciplex with Tyr^<69>, which is located closely to Trp^3 at the interfacial recognition site of the enzyme. On the other hand, the mutant enzyme replaced with phenyl-azophenylalanine in a trans form lost the enzymatic activity. But, photo-illumination induced isoーmerization of the azo group in the enzyme, and the mutant recovered the enzymatic activity slightly. The observed activity is ascribed to increase of alpha-helical conformation of the enzyme accompanied by the isomerization of the azo group from a trans to cis form. Therefore, we have succeeded in preparation of mutant enzymes, in which the activity can be controlled by Photo-illumination. In these cases, Photo-illumination is considered to change the conformation of the enzyme, resulted in alteration of the substrate binding. Less
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