標題: 研究特定胺基酸變異對二氫葉酸還原酶複合態親和性的影響
Studying the key residues affecting the ligand binding affinity of Dihydrofolate reductase.
作者: 黃振華
高雅婷
Huang, Chen-Hua
Kao, Ya-Ting
生物資訊及系統生物研究所
關鍵字: 二氫葉酸還原酶;配體結合親和性;螢光消光;點突變;解離常數;酵素;Dihydrofolate reductase;ligand binding affinity;fluorescence quenching;point mutation;dissociation constant;enzyme
公開日期: 2016
摘要: 二氫葉酸還原酶 DHFR其主要功能以NADPH作為輔酶,將受質二氫葉酸DHF催化還原成四氫葉酸THF,而四氫葉酸THF是去氧胸腺核苷酸(dTMP)合成的前驅物。 DHFR 常作為抗腫瘤及抗菌藥物的標的蛋白,是生物體維持生理功能的酵素。許多文獻指出DHFR突變種具有潛在改變分子動態、配體結合的能力以及酵素活性。 為了觀察造成這些差異的不同,我們利用點突變的方式對特定胺基酸進行取代,並產出突變種M42F及T113V DHFR。實驗中將利用分子本身的光學特性,測得不同酵素—配體複合態的解離常數。 於野生種DHFR的研究,酵素與配體的解離常數分別為NADPH (0.89 μM)、DHF (4.1 μM)、THF (12.5 μM) 以及抑制劑MTX (0.028 μM)。由於本研究中的野生種DHFR在蛋白質的N端加上了含有6個重複的histidine-tag 與19個胺基酸的biotin binding site序列,而造成蛋白質結構上與以往研究所使用的野生DHFR的構型有差異,導致不同配體下的解離常數有明顯的不同。於Met-42 (M42F) 突變種的研究,酵素與配體的解離常數分別為NADPH (33.9 μM)、DHF (63.3 μM)、THF (79.8 μM) 以及抑制劑MTX (0.34 μM)。我們推論是由於並當Met-42 位點置換成更具立體障礙的Phe,擠壓了βB-βC進而影響了helix αC的轉動與移動,使得酵素與輔酶及受質親和性下降;亦推擠到helix αC並使得受質結合位變小,使得酵素與產物親和性下降。於Thr-113 (T113V) 突變種的研究,酵素與配體的解離常數分別為NADPH (20.8 μM)、DHF (65.0 μM)、THF (93.3 μM) 以及抑制劑MTX (0.37 μM)。根據本研究結果,我們推想由於Val與Thr胺基酸具有類似大小,所以對受空間障礙與推擠影響較不顯著。然而,Val-113於殘基上少了氧原子,卻會直接破壞與Asp-27及水分子405的氫鍵網路,進而使得親和性下降。 因此,我們可以觀察到在此突變種下,相同結合位的不同配體其親和性會具有一致改變的現象。我們利用點突變的方式產出突變種M42F及T113V DHFR,實驗觀測不同與配體的複合態親和性會受空間立體障礙或是氫鍵網絡破壞的影響而改變。
The function of dihydrofolate reductase (DHFR) is to catalyze the reduction of dihydrofolate (DHF) to tetrahydrofolic acid (THF) with NADPH as a coenzyme. The catalytic product THF is essential for amino acid catabolism and DHFR is the only enzyme in cells that synthesizes THF. Therefore, DHFR has been recognized as a target for anticancer drugs such as methotrexate (MTX) and the antibacterial agent trimethoprim (TMP). Because of its pharmacological importance, dihydrofolate reductase has been studied extensively. Numerous literatures point out that DHFR mutants have the potential to alter molecular dynamics, ligand binding capacity, and enzymatic activity. We designed point mutations to replace specific amino acids, generated mutant M42F and T113V DHFR and further followed the fluorescence quenching and Förster resonance energy transfer to determine the dissociation constants (KD) of different enzyme-ligand complexes. In the wild DHFR, the enzyme and ligand dissociation constants (KD) were NADPH (0.89 μM), DHF (4.1 μM), THF (12.5 μM), and the inhibitor MTX (0.028 μM), respectively. While compared with previous studies, the wild-type DHFR in our studies was designed with additional 6 repeats of histidine-tag and 19 amino acid biotin binding site sequences at the N-terminus of the protein, resulting in differences in dissociation constants for different ligands. In M42F mutant, the enzyme and ligand dissociation constants (KD) were NADPH (33.9 μM), DHF (63.3 μM), THF (79.8 μM), and the inhibitor MTX (0.34 μM), respectively. We concluded that the stereo-hindrance Phe residue squeezes and affects the rotation and movement of helix αC, resulting in weaken the enzyme-ligand affinity. In T113V mutant, the enzyme and ligand dissociation constants (KD) were NADPH (20.8 μM), DHF (65.0 μM), THF (93.3 μM), and the inhibitor MTX (0.37 μM), respectively. Since the Val and Thr residues are similar in size, so this mutation does not create significant space disturbance. However, Val-113 lacks the oxygen atom in the residue. We concluded that it directly destroys the hydrogen bonding network with Asp-27 and water molecule 405, which decreases the enzyme-ligand affinity.
URI: http://etd.lib.nctu.edu.tw/cdrfb3/record/nctu/#GT070257202
http://hdl.handle.net/11536/140163
Appears in Collections:Thesis