基于嫩度的关键磷酸化糖酵解酶确证及其活性调控机理

Meat tenderness after slaughter and the quality of products is affected by glycolysis directly. Studies in our lab have demonstrated that many kinds of glycolytic enzyme can be phosphorylated, and the rate of glycolysis was affected by phosphorylated glycolytic enzyme. But which is the key phosphorylated glycolytic enzyme and its regulation mechanism of activity is still unknown. This project intends to identify glycolytic enzyme with different phosphorylation level from high tenderness group and low tenderness group using two-dimensional electrophoresis, fluorescence staining and mass spectrometry，confirm the key phosphorylated glycolytic enzyme via the changes of glycolytic enzyme activity and the rate of glycolysis in different phosphorylation level; confirm phosphorylation sites of the key glycolytic enzyme using TiO2 concentration, high performance liquid chromatography and mass spectrometry coupled techniques, identify phosphorylation domain of the key glycolytic enzyme using Fourier transform infrared spectroscopy, circular dichroism spectrum and X-ray diffraction. Analysis the movement of the active site, the change of the reaction force, and the difference of the main structure domain in different phosphorylation level, then discuss the relationship between structure change and glycolytic enzyme activity. The regulation mechanism of phosphorylation on the key glycolytic enzyme activity can be clarified according to the findings of this project. This project will enrich the theory of glycolysis, and provide the theoretical foundation for the research of glycolysis control technology.

糖酵解直接影响畜禽宰后肌肉嫩度和加工品质，本项目组前期研究发现：多种糖酵解酶均可发生磷酸化，磷酸化糖酵解酶影响糖酵解的速率和程度，但哪些磷酸化糖酵解酶是关键酶及其磷酸化调控酶活的内在机理尚不明确。本项目以嫩度差异显著的羊肉为试验材料，采用双向电泳、质谱分析等技术，分离、鉴定差异显著的磷酸化糖酵解酶，改变差异糖酵解酶的磷酸化水平，研究其酶活变化及其对糖酵解速率的影响，确证影响糖酵解的关键磷酸化糖酵解酶；采用二氧化钛富集、高效液相色谱、圆二色谱等技术，检测关键磷酸化糖酵解酶的磷酸化位点，定位关键磷酸化糖酵解酶的磷酸化结构域，解析不同磷酸化水平下活性位点的移动，α-螺旋、β-转角、氢键、范德华力的变化，以及主要结构域构象的差异，探讨这些变化与酶活的关系，揭示关键磷酸化糖酵解酶调控糖酵解酶活性的分子机理，丰富和发展肌肉糖酵解理论，为糖酵解调控技术研发奠定理论基础。

糖酵解直接影响畜禽宰后肌肉嫩度和加工品质，多种糖酵解酶均可发生磷酸化，且磷酸化糖酵解酶影响糖酵解的速率和程度。本研究通过对糖酵解速率差异的样品进行磷酸化蛋白质组学分析，阐明了蛋白质磷酸化参与宰后糖酵解的规律，肌浆蛋白质磷酸化与pH值、糖原含量负相关，与乳酸含量正相关。进一步确证了糖酵解速率差异的肽段和蛋白质，筛选出24个糖酵解速率差异蛋白，聚类在糖酵解和肌肉收缩功能上，明确了差异磷酸化蛋白的磷酸化位点，丙酮酸激酶Thr157，烯醇酶Ser176、Ser177，磷酸果糖变位酶Ser402的磷酸化与糖酵解速率显著相关，说明蛋白质磷酸化在宰后早期可能通过调节参与糖酵解和肌肉收缩的蛋白质间接影响糖酵解途径。对丙酮酸激酶Thr157位磷酸化前后的结构和功能进行分析，发现磷酸化后的丙酮酸激酶结构更为松散，且同样位相对稳定的蛋白结构。为了验证所得的结果，并进一步探讨蛋白质磷酸化调控糖酵解速率的机理，对宰后羊肉添加了激酶抑制剂和磷酸化抑制剂调节蛋白质的磷酸化水平，具有高磷酸化水平的磷酸酶抑制剂组，乳酸含量高，pH值低，确证了蛋白质磷酸化在宰后糖酵解过程中的作用。通过检测糖原磷酸化酶、丙酮酸激酶和磷酸果糖激酶的酶活发现，蛋白质磷酸化正向调节糖原磷酸化酶、丙酮酸激酶和磷酸果糖激酶的活性，进而促进糖酵解的发生；蛋白质磷酸化通过调控酶的活性，间接调控糖酵解，在宰后糖酵解过程中发挥一定作用，初步明确了蛋白质磷酸化通过酶活调控糖酵解速率的机制。