植物螯合素合成酶催化機制研究

Abstract

植物螯合素合成酶 (PCS, EC 2.3.2.15) 利用穀胱甘肽 (GSH) 當作基質進行 植物螯合素 (PCs) 合成。根據先前已發表論文得知 PCS 在生物體內是處於持續表現狀態，因此推測其調控機制可能經由後轉譯修飾。的確，將阿拉伯芥 PCS 序列 (AtPCS1) 經軟體分析後得知其上包含數個可能受到磷酸化修飾位置。而 In vitro 實驗也證明 PCS 活性會受到 casein kinase 2 (CK2) 磷酸化與 calf intestine alkaline phosphatase (CIAP) 去磷酸化調控。當 PCS 被 CIAP 去磷酸化後活性會下降，再經 CK2 磷酸化後則活性恢復。經由放射性點突變實驗證明 AtPCS1 磷酸化位置就在 Thr49。當 Thr49 突變成 Ala 後，AtPCS1 即無法被 CK2 磷酸化，而且活性也明顯下降。另外利用軟體所預測 AtPCS1 3D 立體結構顯示 PCS的 Arg183 在空間位置上非常接近 Thr49，將 Arg183 突變成 Ala 後發現 AtPCS1 仍然可以被 CK2 所磷酸化，但是活性卻明顯下降，推測磷酸化 Thr49 與 Arg183 兩者間可能會相互作用來形成 second substrate-binding site。另一方面，本文亦發現 AtPCS1 磷酸化必須要有 Cd 存在，而且此磷酸化現象會受到基質 GSH 抑制。若是單以 AtPCS1 N 端催化活性區重組蛋白 (AtPCS1-N) 進行相關實驗，發現其活性受到 Cd 與磷酸化的影響更加明顯，但是 AtPCS1-N 磷酸化卻不受 Cd 或 GSH 影響，因此推論 AtPCS1 C-domain 對於 Cd 結合與磷酸化調控佔有重要地位。除此之外，經由酵素動力學實驗結果顯示對於基質 GSH 而言，AtPCS1呈現出 S 形曲線 (sigmoidal curve)，而AtPCS1-N 呈現的則是典型 Michaelis-Menten 動力學圖形 (hyperbolic curve)。再經原態分子量測定發現 AtPCS1 為二元體，AtPCS1-N 為單元體，這些結論都強烈指出 C-domain 可能參與 AtPCS1 四級結構的形成，並且在蛋白質磷酸化反應與活性調控部份扮演重要角色。

Phytochelatin synthase (PCS, EC 2.3.2.15) uses glutathione (GSH) as its substrate to catalyze the synthesis of heavy metal-binding peptides, known as phytochelatins (PCs). PCS has been described as a constitutive enzyme that may be controlled by post-translational modifications. Indeed, we found that the amino acid sequence of PCS from Arabidopsis (AtPCS1) contains several protein phosphorylation motifs. In vitro experiments demonstrate that PCS activity is increased following phosphorylation by casein kinase 2 (CK2), and it decreases following treatment with alkaline phosphatase. Site-directed mutagenesis at several amino acids on AtPCS1 indicates that Thr 49 is the site for phosphorylation; consistent with this idea, the mutant AtPCS1(T49A) cannot be phosphorylated, and its activity is significantly lower than that of the wild-type enzyme. In the predicted three-dimensional structure of AtPCS1, Arg 183 is close to Thr 49. The mutant AtPCS1(R183A) can be phosphorylated, but it shows much lower catalytic activity than the wild-type protein. Interestingly, a second substrate-binding site forms as a result of the interaction of these two amino acids. We propose that Arg 183 interacts with the phosphorylated Thr 49 residue to give the active site of PCS a distinctive shape. Furthermore, the phosphorylation of AtPCS1 by CK2 is dependent on Cd and is inhibited by GSH. The N-terminal catalytic domain of AtPCS1 was expressed (AtPCS1-N), and its catalytic activity was found to be even more sensitive to Cd or phosphorylation status than to that of the full-length enzyme. However, unlike AtPCS1, AtPCS1-N phosphorylation is not controlled by Cd or GSH, indicating a role for the C-domain in Cd-binding and regulation of phosphorylation. In addition, kinetic studies showed that the full-length AtPCS1 activity has a sigmoidal dependence on GSH, and it is estimated to be a dimer, whereas AtPCS1-N was present only in monomeric form. The C-terminal domain may thus participate in the formation of the quaternary structure of AtPCS1, and it may play a regulatory role in protein phosphorylation, forming a competent active site that can accommodate its substrates.