Daquinag A C, Sato T, Koda H, Takao T, Fukuda M, Shimonishi Y, Tsukamoto T
Department of Applied Biological Sciences, Saga University, Japan.
Biochemistry. 1999 Feb 16;38(7):2179-88. doi: 10.1021/bi9819834.
Phenoloxidase inhibitor (POI), found in the hemolymph of housefly pupae, is a novel dopa-containing and cystine-rich peptide that competitively inhibits phenoloxidase with a Ki in the nanomolar range. [Tyr32]POI is a potential precursor molecule also found in the hemolymph that may be posttranslationally oxidized to the dopa-containing peptide after creation of a rigid structure. By employing both a solid-phase peptide synthesis system based on a 9-fluorenylmethoxycarbonyl strategy and a specific air oxidation technique to ensure correct folding, we have been able to synthesize [Tyr32]POI. The synthetic [Tyr32]POI was confirmed to be identical to the native [Tyr32]POI by coelution high-performance liquid chromatography analysis and by enzymatic analysis using the phenoloxidase inhibition assay. To determine the disulfide pairings within the peptides, a series of enzyme hydrolyses and partial reduction/alkylation steps were performed. Three cystine pairs (Cys11-Cys25, Cys18-Cys29, and Cys24-Cys36) were determined by identification of the resulting peptides. The disulfide pairings of the two adjacent Cys residues (Cys11-Cys25 and Cys24-Cys36) were unambiguously assigned by comparing the derived fragments with the two possible isomers synthesized through a novel disulfide-linking technique. The arrangement of the disulfide bridges in POI was found to be topologically identical to those found for several peptides within the inhibitor cystine knot structural family. Although these peptides share a low primary sequence homology and display a diversity of biological functions, they nonetheless share similarities in their cystine motifs and tertiary structure. The tertiary structure model of POI, which was derived through molecular dynamics and energy minimization studies using restraints with determined disulfide connectivities, suggests that POI is a new class member of the inhibitor cystine-knot structural family.
酚氧化酶抑制剂（POI）存在于家蝇蛹的血淋巴中，是一种新型的含多巴且富含胱氨酸的肽，它能以纳摩尔范围内的Ki值竞争性抑制酚氧化酶。[Tyr32]POI也是一种潜在的前体分子，同样存在于血淋巴中，在形成刚性结构后可能会在翻译后被氧化为含多巴的肽。通过采用基于9-芴甲氧羰基策略的固相肽合成系统和特定的空气氧化技术来确保正确折叠，我们成功合成了[Tyr32]POI。通过共洗脱高效液相色谱分析以及使用酚氧化酶抑制试验的酶促分析，证实合成的[Tyr32]POI与天然的[Tyr32]POI相同。为了确定肽内的二硫键配对，进行了一系列酶水解和部分还原/烷基化步骤。通过鉴定所得肽确定了三对胱氨酸（Cys11-Cys25、Cys18-Cys29和Cys24-Cys36）。通过将衍生片段与通过新型二硫键连接技术合成的两种可能异构体进行比较，明确确定了两个相邻半胱氨酸残基（Cys11-Cys25和Cys24-Cys36）的二硫键配对。发现POI中二硫键桥的排列在拓扑结构上与抑制剂胱氨酸结结构家族中的几种肽相同。尽管这些肽的一级序列同源性较低且具有多种生物学功能，但它们在胱氨酸基序和三级结构上仍有相似之处。通过使用确定的二硫键连接性的约束条件进行分子动力学和能量最小化研究得出的POI三级结构模型表明，POI是抑制剂胱氨酸结结构家族的一个新成员。
