標題: 研究多點胺基酸變異對二氫葉酸還原酶複合物之親和力的影響
Studying the Effects of Multiple-sites Mutations in Ligand Binding Affinity of E. Coli Dihydrofolate Reductase
作者: 陳允文
高雅婷
Chen, Yun-Wen
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;反應動力學;酵素動態;解離常數;螢光共振能量轉移;dihydrofolate reductase (DHFR);reaction kinetics;enzyme dynamics;dissociation constant;Förster resonance energy transfer (FRET)
公開日期: 2017
摘要: 本研究選擇一種已經被廣泛研究且有多種結晶結構的酵素——二氫葉酸還原酶(DHFR)作為研究的模型,二氫葉酸還原酶是一種將二氫葉酸(DHF)還原為四氫葉酸(THF)的酵素,並以菸鹼醯胺腺嘌呤二核苷酸磷酸(NADPH)作為輔酶及氫負離子給體。二氫葉酸還原酶是生物體中唯一可以合成四氫葉酸的酵素,而四氫葉酸是去氧胸腺核苷酸(dTMP)合成的前驅物,若dTMP不足會影響DNA合成影響細胞正常表現,是生物內重要的酵素之一。 酵素催化是一個動態過程,酵素經由輔酶的協助將基質催化為產物的過程中,酵素的構象(conformation)通常會隨之發生變化。以定點突變的方法將特定殘基位置以其他氨基酸取代造成酵素構象的改變,藉由酵素與配體的光學變化特性測得酵素-配體解離常數及反應吸收光譜,以作為了解酵素結構與催化功能關係的線索。 本研究中點突變點位選擇分為兩種,其一,選擇Met42、Thr113與Gly121,這些點位是DHFR氨基酸序列上高度保守的位置,且不直接與輔酶或基質作用,避免因為作用力改變直接影響酵素活性,分析上述點位單點與雙點突變的解離常數及自由能變化,結果顯示酵素-輔酶、酵素-基質、酵素-產物三者的親和力會影響反應的過程與結果,而突變自由能變化符合熱力學關係,且雙點突變間的氨基酸點位有交互作用會影響酵素-配體複合物結構;其二,利用點突變在酵素內部產生雙硫鍵以限縮酵素模體的動態,突變種DHFR-W30C/T113C與DHFR-M42C/W47C分別限縮pABG結合袋口兩側helix αB與helix αC的動態,結果顯示兩種突變種都顯著的改變酵素與配體的親和性與系統中反應發生的速度。 本文之DHFR的N端設計有純化使用的6個組胺酸以及19個胺基組成的專一結合生物素(biotin)序列,未來可與基材結合進行螢光相關顯微鏡(fluorescence correlation spectroscopy;FCS)結合單體追蹤技術更深入探索酵素動態與催化反應之奧妙。
We choose oneubiquitousand well-studied enzymes, dihydrofolate reductase (DHFR) as the model in this research. DHFR reduces dihydrofolic acid (DHF) to tetrahydrofolic acid (THF) through hydride transfer by using nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor. THF is an essential precursor for thymidylate synthesis in cells, therefore, lack of DHFR will defect the synthesis of nucleic acid. Moreover, DHFR is known as a target of anti-cancer drugs. It is one of the most important enzymes in biological system. Enzyme catalysis is a dynamic process. Enzymes catalyzing substrates through cofactor usually accompanies conformational changes of enzymes.Mutations of enzymes are designed to alter the ability of the active site and reveal the effect of local residue conformational perturbations of DHFR. We take advantage of fluorescent variationof DHFR and its ligands to obtain the enzyme-ligand complex dissociation constant. And we use the time correlation absorptionspectrum to track the reaction process. We can use those experimental results as clues to discovering the roles of those conformations in catalytic function. We use two strategies for point mutation design. Firstly, we choose residues at highly conserved positon Met42, Thr113 and Gly121 to carry out single and double mutation.We analyze thedissociation and free energy changes of enzyme-cofactor, enzyme-substrate and enzyme-product complex respectively to figure out that the affinity differences of each complex affect the reaction process and that mutation free energy changescomply with thelaw of thermodynamics. Secondly, We design disulfide-bridged mutation speices DHFR-W30C/T113C and DHFR-M42C/W47C to restrict the motion of pABG cleft. Through this experiment, we find that both two mutations alter the affinity of complex and reaction process. DHFR in our study isattached with 6 histidine and 19 residuesspecific binding sequences (biotin). For further study, the biotin sequence can bind DHFR to specific binding site fixed on commercial substrates in order to experiment with fluorescence correlation spectroscopy (FCS), microscopy technique and single molecular technique to questmysteries of enzyme dynamics and catalytic mechanism.
URI: http://etd.lib.nctu.edu.tw/cdrfb3/record/nctu/#GT070357215
http://hdl.handle.net/11536/140577
Appears in Collections:Thesis