液泡焦磷酸水解酶 (簡稱 V-PPase; EC 3.6.1.1) 是一個同雙次體(homodimer)的氫質子轉送酶,具有維持膜內外氫離子濃度梯度的能力,是由一個約80kDa 的多肽鏈組成。許多的結果證明V-PPase 的C 端在許多高等植物中具有較高的同源性且靠近酵素活性催化位置。然而,C 端的真正功能尚未清楚。之前的實驗利用C端切除研究,發現當切除最後5個胺基酸時酵素會明顯地消失活性,但是再額外移除5個胺基酸時,酵素活性卻又恢復至野生株活性的60-70%左右。所以在這次實驗中,我們利用胺基酸定點突變法逐一檢驗V-PPase C端胺基酸的結構及特性。我們的實驗結果顯示 F755A, G759A, F763A, I765A 的酵素活性、質子運送能力、及酵素耦合能力都受到影響降低。 F755A, G759A 的耦合能力只剩下大約野生株的50%。實驗中鉀離子會促進酵素活性,而鈣、氟、鈉 則會抑制V-PPase. 我們的實驗也顯示C端最後10個胺基酸對於各種離子的反應是敏感的。實驗中野生株的V-PPase的酵素 KM 和 Vmax 分別是337 μM 和104 μmol PPi/ mg protein．h,而C端的突變株則明顯的降低了KM 和Vmax 的數值,這也表示我們所產生的突變改變了酵素本身的活性。根據一系列的實驗,我們推測C端胺基酸在酵素活性及質子運輸上扮演舉足輕重的角色。

Vacuolar proton pumping pyrophosphatase (V-PPase; EC 3.6.1.1), a homodimeric proton-translocase sustaining the proton gradient across the membrane, contains a single polypeptide with a molecular mass of approximately 80 kDa. Evidence demonstrated that the C-terminus of V-PPase is relatively conserved in various higher plants and locates near to the catalytic site. However, the real function of C-terminal region is unknown. Our previous study indicated using truncated variants that depletion of last 5 amino acids in C-terminus significantly decreased its activity; however, further removal of next 5 amino acids recovered its activity to about 60-70% of wild type. Accordingly, we identified individual amino acids on C-terminus using site-directed mutagenesis technique that focused on their roles in the structure and function of V-PPase. Our results showed that F755A, G759A, F763A, I765A decreased enzymatic activities, proton translocation, and coupling ratio of V-PPase. Coupling ratios of F755A, G759A retained only about 50% compared to wild type. K+ is stimulatory but Ca2+, F-, and Na+ are inhibitory to the V-PPase. Our results suggested that these C-terminal amino acids are sensitive to various ions especially last 10 residues. For wild type V-PPase, KM and Vmax are about 337 □M and 104 □mol PPi/ mg protein．h, respectively. Mutation of C-termianl amino acids apparently decreased their values of KM and Vmax, indicating a change in their enzymatic reactivity. In conclusion, C-terminal region of plant V-PPase would play an important role on enzymatic mechanism and participate in enzymatic and proton translocation reactions.

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