溶液酸鹼值對人類 Pin1 穩定性及含脯胺酸衍生物之基質對其酵素活性效率影響

Study of Human Pin1: pH-dependent Stability and the Impacts of 4-Substituted Proline Derivatives on the Catalytic Efficiency

吳淑華(Hsu-Hua Wu)

指導教授 ： 洪嘉呈

國立清華大學/理學院/化學系/碩士(2014年)

摘要

人類 Pin1 蛋白質是細胞分裂的調節蛋白之一，可調控含有被磷酸化的絲胺酸或者蘇胺酸與脯胺酸之間的胜肽鍵進行順反異構，進而影響體內蛋白質的折疊，目前發現阿茲海默症的成因之一與 hPin1 的功能調控有關，為了避免疾病的發生或是設計其治療藥物，我們必須先了解酵素催化的反應機構。
我們利用大腸桿菌表現的方式大量得到目標蛋白，並且用溫和的純化方法保持其活性。在 CD 變溫實驗當中，得知 hPin1 在 pH 7-8 之間具有良好的結構穩定性，且隨著降低水溶液的 pH 值，會改變蛋白質的二級結構與穩定性。將基質 Ac-ApTPY-pNA 上的脯胺酸用 (2S,4S)-hydroproline (hyp)，(2S,4R)-
hydroproline (Hyp)，(2S,4S)-fluoroproline (flp)，(2S,4R)-fluoroproline (Flp) ，(2S,4S)-methoxyproline (mop)，(2S,4R)-methoxyproline (Mop)，(2S)-4,4-difluroproline (Dfp)，(2S)-4-ketoproline (Kep) 這些非天然的胺基酸衍生物進行取代，利用 Protease-
coupled assay 觀察構形對基質結構與 hPin1 催化效率的影響。從結果上來看，在 LiCl/TFE 的環境中，用 Kep 取代的胜肽其 pT-Xaa 胜肽鍵 cis 式比例提高；而用Hyp、hyp 與 Dfp 取代的胜肽鍵 cis 式比例降低。而 hPin1 對用 flp、Flp、Mop 與 Dfp 取代的基質有較好的催化效率，對用 Kep 取代的基質其催化效率在本研究中所使用受質中表現最差，而其他變異基質則與原胜肽無明顯變化。由此可以看出立體電子效應並不是造成不同環構形對 pT-Xaa 之間之胜肽鍵順反式的比例，與影響到 hPin1 催化效率的主要原因。
目前對於 hPin1 催化反應的途徑仍不清楚，但是從我們的研究當中發現藉由修飾脯胺酸 Cγ 位置對 hPin1 催化效率有明顯的影響且其原因與立體障礙和疏水作用力有關，希望此結果有助於進一步了解該酵素的催化反應機構，並在設計藥物上有所幫助。

關鍵字

順反異構酶 ； 脯胺酸 ； 催化效率 ； 胜肽

並列摘要

Human Pin1, a peptidyl-prolyl isomerase catalyzes the cis/trans isomerization of specifically phosphothreonine/phosphoserine-proline peptide bond, and it is used as molecular switches to regulate cell cycle progression to affect biological activity of protein. Recently it has been found that Alzheimer’s disease may be associated with the dysfunction of human Pin1. In order to avoid the occurrence of the disease or design the drug for treatment, we have to understand the isomerization mechanism of hPin1.
We used E.coli expression system to prepare hPin1
, and maintained its bioactivity by a moderate purification method. Thermal unfolding measurements indicate hPin1 has a high conformational stability between pH 7 and pH 8, and lowering the pH value will destabilize the protein. We replaced the proline residue in the substrate Ac-ApTPY-pNA with (2S,4S)-hydroproline (hyp), (2S,4R)-hydroproline
(Hyp), (2S,4S)-fluoroproline (flp), (2S,4R)-fluoroproline (Flp), (2S,4S)-methoxy-proline (mop), (2S,4R)- methoxyproline (Mop), (2S)-4,4-difluroproline (Dfp), (2S)-4-ketoproline (Kep) to change the ring pucker preference and study the consequence on catalytic efficiency of hPin1. As determined by a protease-coupled assay, replacement of proline with Kep induces a deceased cis content. The cis content of the Hyp, hyp, Dfp peptide was increase upon replacing proline into these derivatives. hPin1 exhibits a better catalytic efficiency toward the flp, Flp, Mop, Dfp peptide than the Pro peptide, and has a lowest catalytic efficiency to the Kep-containing peptide. The results show that a pre-organized ring pucker may not be the key factor contributing to the cis/trans isomer ratio of the peptide and the catalytic efficiency of hPin1.
Although the details of the hPin1 catalytic mechanism of cis/trans isomerization is not yet clear, we find that the impacts of the 4-substituded proline derivatives on the catalytic efficiency of hPin1 may be related to steric and hydrophobic effects. Our results may be useful for understanding the catalytic mechanism and the substrate specificity of hPin1.

並列關鍵字

pin1 ； catalytic efficiency ； proline

參考文獻

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