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Photoresponsive Membrane-Associated Mutant Enzyme Prepared by Semisynthesis

半合成光响应膜相关突变酶

基本信息

批准号：
01470112
负责人：
IMANISHI Yukio
金额：
$ 4.16万
依托单位：
Kyoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1989
资助国家：
日本
起止时间：
1989 至 1990
项目状态：
已结题

项目摘要

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 … More 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

将芳香族非天然氨基酸掺入磷脂酶A_2中，研究了光照对酶活力的影响。磷脂酶A_2中的Trp^3是磷脂与分子间的界面识别位点，它将膜组装体中的磷脂与分子分散的溶液区分开来，用半合成法制备了磷脂酶A_2的突变酶，其中Trp^3被萘丙氨酸、蒽丙氨酸和苯偶氮苯丙氨酸取代。其制备过程如下：i）选择性地对<epsilon>磷脂酶A_2上的赖氨酸残基的^ -氨基进行酰胺化，ii）通过Edman降解（DES 3）从N-末端去除连续的三个氨基酸，iii）通过常规液相合成制备Boc-Ala-Leu-Xaa-OH（Xaa代表芳香族非天然氨基酸），iv）将三肽与DES 3偶联，v）去除Boc基团，并纯化突变酶。酶活测定采用紫外分光光度法 ...更多信息以含[13 <14>C]标记二棕榈酰磷脂酰胆碱的脂质体为底物。与天然磷脂酶A_2相比，被萘丙氨酸或蒽丙氨酸取代的突变酶的活性很低。光照射进一步降低了酶活性。推测磷脂酶A_2中掺入的非天然氨基酸可能与Tyr^形成激基复合物<69>，Tyr ^位于该酶界面识别位点上靠近Trp^3的位置。另一方面，用反式形式的苯基偶氮苯丙氨酸取代的突变酶失去了酶活性。但光照诱导酶中偶氮基团发生异构化，突变体酶活性略有恢复。所观察到的活性归因于伴随着偶氮基团从反式异构化为顺式形式的酶的α-螺旋构象的增加。因此，我们已经成功地制备了突变酶，其中的活性可以通过光照来控制。在这些情况下，光照射被认为改变了酶的构象，导致底物结合的改变。少