Photoresponsive Membrane-Associated Mutant Enzyme Prepared by Semisynthesis

芳香非自然氨基酸在磷脂酶A_2中被吸收，以及光照明对酶活性的影响已经被研究。磷脂酶A^2中的Trp^3已知固定在表面识别站点上，其中磷脂酶A_2中的表达表达在分子分散的溶液中。磷脂酶A_2的突变酶由半合成制备，其中Trp^3被替换为naphylalanine、anthylalanine和phenylazophenylanine。制备过程的大纲如下： i）<epsilon>Lys保留者中的氨基组中的磷酸酶A_2的胺组的胺酸选择; ii）Edman脱氨（DES 3）对N-末端的三种成功氨基酸的刺激; iii）对Boc-Ala-Leu-Xaa-OH（Xaa表现出芳香非天然氨基酸）的常规液体相合成的制备; iv）与DES 3的三肽偶联; v）从Boc组中去除和突变酶的纯化。由U分析的酶活性。 ... More sing liposome containing [^<14>C]labeled dipalmitoylphosphatidylcholine as substrate。由naphthylalanine或anthrylalanine替换的突变酶显示了一个非常小的活动，与本机磷脂酶A_2相比较。Photo-Illumination furthermore被缩小到酶体活性。Presummably，在磷脂酶A_2中形成一个与Tyr^的exciplex<69>，将Trp^3定位在酶的界面识别站点。在另一只手上，用苯基-氮杂苯丙氨酸在一个变压器形式上失去了酶活性。但是，光照明诱导的ISO--氮唑组在酶中的合并，并且突变体略微恢复了酶活性。观察性活动被归类为由氮杂群的异构化从反式到顺式形式的α-酶的基本构成。因此，我们成功地准备了突变酶的突变体，在任何活动都可以被照片照明控制的地方。在这些案例中，照片照明被认为是为了改变酶的构成，结果是在底层绑定的改变中。Less（低）

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