△5-3-酮基類固醇異構酵素突變株化學修飾之研究

Abstract

Pseudomonas testoteroni 中發現的酵素 △5-3-酮基類固醇異構酵素 (KSI，EC 5.3.3.1) 能以擴散速率控制，立體選擇及立體保留的方式將 △5-3-酮基類固醇轉變成 △4-３-酮基類固醇。其催化反應中，Tyr-14 扮演 general acid，Asp-38 扮演 general base。當類固醇進到酵素活性區時會對 Tyr-14 的螢光產生淬息現象，但因螢光強度不高而靈敏度不夠，無法使用於晶片檢測上。所以，為了在酵素活性區內修飾一螢光物質以增大其螢光強度，分別以定點突變方式設計了半胱胺酸於胺基酸序列 54、55、82 的位置，再將此突變株與 5,5’-Dithiobis(2-nitrobenzoic acid) (DTNB) 及螢光物質 IANBD ester (I-9) 及 1,5-IAEDANS (I-14) 反應。結果發現 DTNB 可修飾於所引入的半胱胺酸上，而上述兩類螢光物質除了可以修飾在 Cys-54 外，並無法進入酵素活性區深處與 Cys-82 反應。反之，以胜肽圖譜法分析螢光物質所修飾的胺基酸位置發現，I-14 可修飾在胺基酸 Asp-38 上。這是因為 Asp-38 位於酵素活性區疏水環境中親核性變強，以致於可與螢光物質反應的結果。而 I-9 修飾於胺基酸後螢光團會斷裂，導致無法觀測 I-9 的紫外-可見光及螢光變化。以 DTNB 和 I-14 修飾後的 KSI 活性至少降低了 103 倍，而由類固醇結合能力的 KD 值分析，修飾後的 KSI 與類固醇的結合能力變差。 DTNB 修飾在 Cys-82 時，紫外光吸收剩下 55 ﹪，且由 324 nm 藍位移至 318 nm；DTNB 修飾在 Cys-54 時，紫外光吸收剩下 50 ﹪，沒有藍位移現象。I-14 修飾在 Asp-38 上時，螢光由 498 nm 藍位移至 452 nm (Y55F/Y88F) 及 464 nm (Y55F/F82C/Y88F)，螢光強度也分別增強 3.4 倍 及 3.7 倍，而I-14 同時修飾在 Asp-38 與 Cys-54 上時，螢光無藍位移產生，但螢光強度約增強了 6 倍。此外，利用設計在酵素表面的半胱胺酸可使 KSI 與奈米金條反應而固定在奈米金條上，此現象或可應用於開發類固醇檢測試片或晶片。

△5-3-Ketosteroid isomerase (KSI, EC 5.3.3.1) of Pseudomonas testoteroni promotes the highly efficient isomerization of △5-3-Ketosteroids to △4-3- Ketosteroids by a direct and stereospecific transfer of the 4β-proton to the 6-β-position. Asp-38 and Tyr-14 function as the general base and the general acid of the catalytic reaction, respectively. The fluorescence intensity of Tyr-14 can be quenched when enzyme binds with some steroids such as 19-nortestosterone. Since the intrinsic fluorescence intensity of KSI is too weak to apply on biochip for steroid detection, two dyes (I-14 and I-9) are hence selected as labeling agents which are potentially useful for improving fluorescent property of KSI. KSI mutants, Y55F/Y88F, Y55F/Y88F/F54C and Y55F/Y88F/F82C, which are designated as Y14only, 54C, and 82C, respectively, were constructed for labeling study. The dye I-14 can only performed a single-labeling on Y14only and 82C, whereas a double-labeling can be found when 54C was incubated with I-14. Studies from peptide mapping and tandem mass spectrometry of the labeled KSI mutants revealed that both the newly recruited cystein residue and Asp-38 in the mutant can be modified. The modification on Asp-38 can be explained by assuming its unusual nucleophilicity resulting from the highly hydrophobic environment. The outcomes of labeling with I-9 were similar to those of with I-14. However, interestingly, on the I-9 labeled mutants, the corresponding chromophoric moiety can be further hydrolyzed. All of these observations were confirmed both by ESI-MS and UV-spectroscopy analysis of proteins. The fluorescence intensity of I-14-labeled mutants is enhanced, whereas for I-9-labeled mutants, the fluorescence data were not reliable owing to the continuous decomposition of chromophoric group of I-9. Both the catalytic activity and steroid binding ability of mutants labeled with DTNB or I-14 decrease significantly. Further study will be performed on improving the recognition of steroid by this group of KSI mutants.