| Summary: | 碩士 === 國立臺灣師範大學 === 物理學系 === 93 === Thioesterase 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.
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