Abstract
為了探索植物細胞液泡囊膜上一個和能量有關的酵素: 焦磷酸水解酵素 (H+-PPase) 其結構與功能之間的關係，我們利用了 (1) 化學修飾 法 (包括動力學分析及氨基酸定序列法) (2) 生物光譜分析 (包括吸收光 譜 . 螢光光譜及旋光光譜) (3) 物理因子的影響 (包括高壓及熱力學分 析) 等等方法來探求。 我們將 H+-PPase 從白化綠豆下胚軸中純化出來 ，定性分析後得知它是一個由兩個 73 kDa 的次體所組成的均質複體，分 子量為 145 kDa。在化學修飾法中我們選用了一些專一性的藥物與 H+- PPase 作用形成共價鍵，而由動力 學的分析結果指出在此酵素的催化 中心部位含有 aspartic acid、 tyrosine 、 lysine 和 cysteine 等 必須氨基酸。 由氨基酸定序列法得知 aspartic acid 這個必須氨基酸是 位在第 283 的位置，而且是面對 cytosol 的方向。 從高壓的實驗中發 現壓力處理使得 H+-PPase 由雙次體解離成單次體，並且在壓力解除後， 大部分分解後的單次體重組回原來雙次體的形態，然而在此分解及重組的 過程中，次體之間的相互作用使得H+-PPase 的生理活性喪失，而且無法 恢復。 另由熱力學實驗得知，在不同溫度處理下 H+H+-Pase 具有一個 轉換點，並且會受受質 (Mg2+-PPi) 的保護，可見 H+-PPase 不同程度的 改變對熱抑制 會有不同的敏感度。 綜合以上實驗結果，我們提出了 H+- PPase 催化中心的結構圖，以及次體之間分解及重組的機制與作用關係， 並且探討了其熱力學特性。 由這些結果讓我們對H+-PPase 結構與功能之 間的關係有了深入的了解。 Plant vacuolar vesicles contain a novel H+-translocating pyrophosphatase (H+-PPase), which catalyzes both the hydrolysis of PPi and the electrogenic translocation of H+ from the cytosol to the lumen of the vacuole. The H+-PPase was purified by two-step detergent solubilization and chromatography from etiolated hypocotyls of mung bean (Vigna radiata L.). The vacuolar H+-PPase from mung bean is a homodimer and the molecular mass was 145 kDa for the native enzyme. To investigate the relationship between structure and function of this H+- translocating enzyme, several approaches were used: (1) chemical modification (kinetics and peptide mapping), (2) biological spectroscopy (absorption, fluorescence, circular dichroism, and x-ray diffraction), (3) physical factors (high hydrostatic pressure and thermodynamic analysis). In chemical modification, we suggested that one essential copy of carboxylate, tyrosine, lysine, and cysteine residues are involved in the catalytic function,probably at the catalytic site of vacuolar H+-PPase. The exact site of the essential carboxylate residue was identified with peptide mapping. The results of high hydrostatic pressure indicate the importance of protein-protein interaction for the enzymatic activity of this proton translocating enzyme. The inactivation models were proposed to construe the pressure inactivation of vacuolar H+-PPase. Thermodynamic study indicated that two distinct states of H+-PPase were induced by heat and exhibited different transition temperature in the presence and absence of ligands. By these approaches, we were able to reveal the relationship between the structure and function of vacuolar H+-PPase. A tentative structural mapping of the active site and the working models about the subunit interactionof this homodimer enzyme were proposed. The thermodynamic properties of H+-PPase on the stability of enzyme structure and function were discussed.