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dc.contributorTai-Huang Huangen_US
dc.contributor.authorChing-Yu Chouen_US
dc.description.abstract大腸桿菌硫酯(TEP-I)在生物上的功能是幫助醯基輔水解分裂硫酯。我們所研究的酵素除了是一種硫酯之外,它也屬於絲蛋白中SGNH水解中的一支。其中和催化過程有直接相關的的胺基酸可分為催化三元素(Ser10, Asp154, His157)和氧陰離子洞(Ser10和Asp154骨架上的氨基,和Asn73末端的氨基)。為了研究酵素催化過程,我們所取用的反應物為DENP( diethyl p-nitrophenyl phosphate)。因為DENP和TEP-I的反應過程包括酵素催化過程中的中間態(MC)和過度時期(TC)。我們利用二維的異核核磁共振脈衝序列,在靜磁場14.7T下來量測自旋-晶格遲緩速率(R1)、自旋-自旋遲緩速率(R2)及15N{H}異核交互作用增億參數(NOE)。我們使用二次擴散分析(quadric diffusion analysis)獲得擴散張量(diffusion tensor)及無模型法則(model-free formalism)決定次序參數(S2)、有效相關時間(te)及化學交換參數(Rex);另外,我們進一步地經由簡化的光譜密度的對應(reduced spectral density mapping)得到光譜密度函數。我們也利用了分子模擬計算得到原子階層的圖像,也將其與實驗做了比較分析。 利用上述的計算分析,我們得到了酵素在不同狀態下的動態模擬,了解酵素是如何調整動性減低反應能量。zh_TW
dc.description.abstractThioesterase I (TEP-I) of Esherichia coli catalyzes the hydrolytic cleavage of fatty acyl-coenzyme A (CoA) thioesters. In addition to be a thioesterase, TEP-I has been shown to be a serine protease of the SGNH-hydrolase family. The residues involve in the catalytic process include the catalytic triad of Ser10, Asp154 and His157, and the oxyanion hole groups, which have been identified as the amide groups of Ser10 and Gly44 and the side chain of Asn73. The binding process of TEP-I with its inhibitor DENP (diethyl p-nitrophenyl phosphate) involves a fast formation of the Michaelis-Menten complex (MC) and a subsequent slow formation of the tetrahedral complex (TC). This slow kinetic makes TEP-1 an excellent model system for investigating the molecular structures and dynamics of the catalytic intermediate states. We have determined the backbone 15N NMR spin relaxation rates of the three catalytic states of TEP-I, namely the free enzyme, the TEP-1/DENP Michaelis complex, and the TEP-1/DENP tetrahedral complex at 600MHz (1H frequency). We used the Model-free approach to calculate generalized order parameters, S2, the effective correlation times, e, and a chemical exchange rate, Rex. We found that significant number of NH bonds exhibit observed s-ms time scale motion in the MC state. Changes in the generalized order parameters, characteristics of internal motion, along the catalytic pathway were also observed. His157 and Tyr15, which are located in active site pocket and which were shown by X-ray crystal structure to form - stacking in apo-form, showed significant disorder in the MC state. Furthermore, the mobility of the loop around the binding pocket is also affected by the DENP binding. We have also conducted molecular dynamics simulation of TEP-I of apo-form, MC, and TC. To analyze the overall motion and atomic fluctuation in the two-step catalytic process, we have calculated B-factor, dipolar nuclear relaxation order parameters, and the hydrogen bond network in the neighborhood of the oxyanion hole groups. The B-factor profile of each residue is in generally in good accord with the X-ray result. The 15N NMR nuclear relaxation order parameter indicated that the loop near the catalytic triad Ser10 is mostly disordered in T.S. in nano-pico second time scale. The dynamical characteristic was also confirmed by molecular dynamics simulation. The analysis of hydrogen bond network is aimed at revealing the inter-block motions of different catalytic states. We found that the hydrogen bonds among the neighboring residues of the Asn73 oxyanion hole are rarely formed in the apo-form. Thus, motion within blocks is less mobile in T.C. than apo-form of enzyme and this result is in very good agreement with NMR experiments. In conclusion, we showed that the mobility of catalytic triad plays a crucial role in the catalytic process.en_US
dc.subjectserine proteaseen_US
dc.subjectbackbone dynamicsen_US
dc.subjectmolecular dynamics simulationen_US
dc.titleProtein Backbone Dynamics of the Catalytic Intermediatesof a Serine Protease:A Case Study of Escherichia coli Thioesterase/Protease Ien_US
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