穀胱甘肽/精胺質水解酵素活性探針之設計與開發

穀胱甘肽/精胺質 (glutathionylspermidine, Gsp) 是由一分子穀胱甘肽 (glutathione) 及一分子精胺質 (spermidine) 所組成之生物分子；主要存在於大腸桿菌等少數細菌及單細胞原(protozoa) 中。而Gsp synthetase/amidase (GspSA) 為一負責催化大腸桿菌中Gsp的合成 (同時消耗一分子ATP) 及水解互相拮抗的雙功能酵素；兩個酵素活性分別位於整個蛋白質兩端各自獨立摺疊的活性區塊 (activity domain)。先前的研究也證實大腸桿菌中Gsp amidase屬於半胱胺酸水解酶 (cysteine protease)，以第59號半胱胺酸為催化親核基 (catalytic nucleophile)。根據文獻報導，GspSA 在大腸桿菌氧化還原調控中扮演著重要的角色；GspSA兩個相拮抗之酵素活性，因應氧化壓力的不同而有所改變。藉由活性探針來觀察大腸桿菌中Gsp amidase的存在 (定性) 及含量 (定量)，將有助於我們探討 GspSA調控兩個相反酵素活性的特殊機制。本論文藉由蛋白質晶體結構與酵素反應機制，設計並合成了數個 Gsp amidase 的活性探針 (Activity-Based Probes; 簡稱 ABPs)。在 ABPs 的設計上以γ-Glu-Ala-Gly代替穀胱甘肽的三胜肽 (γ-Glu-Cys-Gly) 作為酵素專一性辨識區；並選擇活化酮基 (activated ketone) 當成特定反應基團，使ABPs可以與 Gsp amidase 中參與催化的半胱胺酸 (Cys59) 形成共價鍵鍵結。並進一步地經由ABPs上的標籤基團 (tag)，可以觀察此酵素的存在 (定性) 及含量 (定量)。在合成 ABPs 的過程中，我們改良了傳統合成策略，由二醇類的化合物作為起始物，合成活化酮基類的活性探針，藉此簡化合成的步驟並提高安全性。最後經由電腦模擬與酵素活性的分析，決定最佳的活性探針，並且成功的利用此活性探針在菌體中標示到 GspS。未來期望可藉由此活性探針進一步去探討 Gsp amidase 在生物體內活性的調控並協助發展出強效活性的酵素抑制劑來治療原蟲疾病的藥物。

關鍵字

穀胱甘肽/精胺質；水解酵素；活性探針

並列摘要

Glutathionylspermidine (Gsp), an amide-bond conjugate of glutathione and spermidine, is mainly found in some protozoal parasites and E. coli. Glutathionylspermidine sythetase/amidase is a bifunctional enzyme with separate activity domains to catalyze both the synthesis and hydrolysis of Gsp. Recently we discovered that Gsp synthetase/amidase plays an important role in redox regulation in E. coli. The amidase activity changes depending on the condition of various oxidative stress. In order to qualitatively and quantitatively monitor the amidase activity level in vivo, it is necessary to design and synthesize specific activity-based probes (ABPs) to investigate how Gsp amidase is involved in redox regulation and how the synthetase and amidase communicate with each other in the oxidative defense. Based on the catalytic mechanism and X-ray structural information of Gsp amidase, the

並列關鍵字

glutathionylspermidine ； protease ； activity-based probes

參考文獻

16. Sachidanandam, R.; Weissman, D.; Schmidt, S. C.; Kakol, J. M.; Stein, L. D.; Marth, G.; Sherry, S.; Mullikin, J. C.; Mortimore, B. J.; Willey, D. L.; Hunt, S. E.; Cole, C. G.; Coggill, P. C.; Rice, C. M.; Ning, Z.; Rogers, J.; Bentley, D. R.; Kwok, P. Y.; Mardis, E. R.; Yeh, R. T.; Schultz, B.; Cook, L.; Davenport, R.; Dante, M.; Fulton, L.; Hillier, L.; Waterston, R. H.; McPherson, J. D.; Gilman, B.; Schaffner, S.; Van Etten, W. J.; Reich, D.; Higgins, J.; Daly, M. J.; Blumenstiel, B.; Baldwin, J.; Stange-Thomann, N.; Zody, M. C.; Linton, L.; Lander, E. S.; Altshuler, D., A map of human genome sequence variation containing 1.42 million single nucleotide polymorphisms. Nature 2001, 409, 928-33.

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穀胱甘肽/精胺質水解酵素活性探針之設計與開發

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