破傷風桿菌之質子傳送焦磷酸水解酶中基要酪胺酸之功能探討

嵌膜性質子傳送焦磷酸水解酶能夠水解焦磷酸，並且利用其水解能量產生跨膜電化學質子梯度。許多研究指出，酪胺酸在各種酵素中扮演數種功能性的角色，包括作為轉譯後調控的磷酸化修飾位、排序信號跨膜蛋白的目標、以及直接參與催化反應。為了瞭解質子傳送焦磷酸水解酶中酪胺酸所扮演的角色，破傷風桿菌質子傳送焦磷酸水解酶中的所有酪胺酸，藉由定點突變技術置換為丙胺酸，並且檢驗其突變後酵素之功能性特性。我們發現其中四個突變株(Y226A, Y392A, Y414A and Y471A突變株)無法正常表現。另外有一株正常表現之突變株Y175，失去其水解焦磷酸之活性。我們進一步針對這五株突變株進行其他種胺基酸的置換。所有酪胺酸置換為苯丙氨酸或是色胺酸的突變株，都能與去半胱氨酸突變株有一樣好的蛋白質表現。另外在這些突變株中，Y414S和Y414T突變株顯現出相較其他突變株高出許多之水解焦磷酸之活性，代表其側鏈上的羥基對於其酵素活性十分重要。更深入的分析離子效應，可發現Y414S和Y414T株突變株擁有不同敏感度的鉀離子以及鈉離子抑制特性。基於這些定點突變結果以及預測之三維結構，我們推測酪胺酸414與白胺酸441形成之氫鍵對於維持鉀離子結合區域的結構十分必要。總結以上結果，此研究發現質子傳送焦磷酸水解酶中的酪胺酸，具有許多未能預期之功能

關鍵字

破傷風桿菌；焦磷酸水解酶；質子；酪胺酸；點突變

並列摘要

Membrane-integral proton-translocating pyrophosphatase (H+-PPase; EC3.6.1.1) hydrolyzes pyrophosphate (PPi) and uses the energy to create the electrochemical proton gradient cross the membrane. Many studies indicated that tyrosine plays several functional roles in variety of enzymes, such as the phosphorylation site of post-translational regulation, the sorting signal of transmembrane proteins targeting, and direct involvement in enzymatic activity. To determine the critical functional roles of tyrosines in H+-PPase, all tyrosine residues from Clostridium tetani E88 H+-PPase were substituted with alanine by site-directed mutagenesis in Cys-less template and their functional properties examined. We found that 4 variants (Y226A, Y392A, Y414A and Y471A mutants) could not be expressed properly. Another mutant with normal expression, Y175A mutant, displayed deterioration in PPi hydrolysis activity. Moreover, we substituted these five tyrosines to other kinds of amino acids. All variants of Tyr → Phe or Tyr → Trp could be expressed as well as Cys-less mutant. Among these mutants, Y414S and Y414T exhibited a higher PPi hydrolysis activities compared to other substitutions, suggesting that the hydroxyl group is essential for the enzymatic activity. Furthermore, Y414S and Y414T mutants showed the distinct sensitivity of K+-stimulation and Na+-inhibition from Cys-less background. Based on the mutagenesis results and predicted structure, we speculate that Tyr-414 formed a hydrogen bond with Leu-441, which is essential for maintaining the structure of K+-binding site. Taken together, several unexpected functions of tyrosine residues in H+-PPase were revealed in this study.

並列關鍵字

Clostridium ； tetani ； H+-pumping ； pyrophosphatase ； tyrosine ； site-directed mutagenesis

參考文獻

1. Maeshima, M. (2000) Vacuolar H+-pyrophosphatase. Biochim. Biophys. Acta 1465, 37-51

2. Rea, P. A. and Poole, R. J. (1993) Vacuolar H+-translocating pyrophosphatase. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, 157-180

3. Serrano, A., Perez-Castineira, J. R., Baltscheffsky, H. & Baltscheffsky, M. (2004) Proton-pumping inorganic pyrophosphatases in some archaea and other extremophilic prokaryotes. J. Bioenerg. Biomembr. 36, 127–133

4. Maeshima, M. (2001) Tonoplast transporters: organization and function. Annu. Rev. Plant Physiol. Plant Mol. Biol. 52, 469–497

5. Belogurov, G. A. and Lahti, R. (2002) A lysine substitute for K+. A460K mutation eliminates K+ dependence in H+-pyrophosphatase of Carboxydothermus hydrogenoformans. J. Biol. Chem. 277, 49651–49654

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破傷風桿菌之質子傳送焦磷酸水解酶中基要酪胺酸之功能探討

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