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。

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.

1. Mark Gerstein, A. M. L., and Cyrus Chothia. (1994). Structural Mechanisms for Domain Movements in Proteins. Biochemistry 33, 6739-6749.
2. Karplus, M. M., J. A. (1983). Dynamics of proteins: elements and function. Annu. Rev. Biochem. 53, 263-300.
3. Andrews, B. K., Romo, T., Clarage, J. B., Pettitt, B. M. & Phillips, G. N. (1998). Characterizing globlal substates of myoglobin. Structure 6, 587-594.
4. Tsou, C. L. (1998). Active site flexibility in enzyme catalysis. Ann. N. Y. Acad. Sci. 864, 1-8.
5. Miller, G. P. B., S. J. (1998). Stretching exercise-flexibility in dihydrofolate reductase catalysis. Chem. Biol. 5, R105-R113.
6. Eyring, H. (1935). Chem. Rev. 17, 65-77.
7. Wolfenden, R. (1969). Nature 223, 704-705.
8. Kass, L. R., Brock, D. J. H. & Bloch, K. (1967). Beta-hydroxyldecanoyl thioesterase I. Purification and properties. J. Biol. Chem.242, 4418-4431.
9. Branes, E. m., Jr & Wakil, S. J. (1968). Studies on the mechanism of fatty acid synthesis. XIX. Preparation and general properties of palmityl thioesterase. J. Biol. Chem. 243, 2955-2962.
10. Pacaud, M. U., J. (1971). Isolation and some properties of a proteolytic enzyme from E. Coli (Protease I). Eur. J. Biochem. 23, 435-442.
11. Spencer, A. K., Greenspan, A. D. & Cronan, J. E., Jr. (1978). Thioesterase I and II of E. Coli. Hydrolysis of native acyl-acyl carrier protein thioesters. J. Biol. Chem.253, 5922-5926.
12. Cho, H. C., J. E., Jr. (1993). E. Coli thioesterase I, molecular cloning and sequencing of the structural gene and identification as a periplasmic enzyme. J. Biol. Chem. 268, 9238-9245.
13. Lee, Y. L., Chen, J. C. & Shaw, J. F. (1997). The thioesterase I of E. Coli is an arylesterase and shows stereospecificity for protease substrates. Biochem. Biophy. Rese. Commu. 231, 452-456.
14. Cho, H. C., J. E., Jr. (1994). Protease I of E. Coli functions as a thioesterase in vivo. J. Bacteriol. 176, 1793-1795.
15. Ichihara, S., Matsubara, Y., Kato, C., Akasaka, K. & Mizushima, S. (1993). Molecular cloning, sequencing, and mapping of the gene encoding protease I and characterization of protease and protease-defective E. Coli mutants. J. Bacteriol. 175, 1032-1037.
16. Yu-Chih Lo, S.-C. L., Jei-Fu Shaw & Yen-Chywan Liaw. (2003). Crystal Structure of Escherichia coli Thioesterase I/Protease I/Lysophospholipase L1: Consensus Sequence Blocks Constitute the Catalytic Center of SGNH-hydrolases through a Conserved Hydrogen Bond Network. J. Mol. Biol. 330, 539-551.
17. Massiah, M. A., Viragh, C., Reddy, P. M., Kovach, I. M., Johnson, J., Rosenberry, T. L., & Mildvan, A. S. (2001). Biochemistry 40, 5682-5690.
18. Cohen, J. A., Oosterbaan, R. A., & Berends, F. (1967). Methods in Enzymology 11, 686-702.
19. Sergiy I. Tyukhtenko, A. V. L., Chi-Fon Chang, Yu-Chih Lo, Shin-Jye Lee, Jei-Fu Shaw,Yen-Chywan Liaw, & Tai-Huang Huang. (2003). Sequential Structural Changes of Escherichia coli Thioesterase/Protease I in the Serial Formation of Michaelis and Tetrahedral Complexes with Diethyl p-Nitrophenyl Phosphate. Biochemistry 42, 8289-8297.
20. Palmer, A. G. I., Rance, M. & Wright, P. E. (1991). Intramolecular motions of a zinc finger DNA-binding domain from Xfin characterized by proton-detected natural abundance 13C heteronuclear NMT spectroscopy. J. Am. Chem. Soc. 113, 4371-4380.
21. Cavanagh, J., Wayne, J. F., Palmer, A. G. III, & Skelton, M. J. (1996). Protein NMR spectroscopy principles and practice.
22. Evan, J. N. S. (1995). Biomolecular NMR Spectroscopy.
23. Abragam, A. (1961). Principles of Nuclear Magnetism, Clarendon Press, Oxford.
24. Lipari, G. S., A. (1982). Model-Free Approach to the Interpretation of Nuclear Magnetic Resonance Relaxation in Macromolecules. 1. Theory and Range of Validity. J. Am. Chem. Soc. 104, 4546-4559.
25. Lipari, G. S., A. (1982). Model-Free Approach to the Interpretation of Nuclear Magnetic Resonance Relaxation in Macromolecules. 2. Analysis of Experimental Results. J. Am. Chem. Soc. 104, 4559-4570.
26. Clore, G. M., Szab, A., Bax, A., Kay, L. E.,Driscoll, P. C. & Gronenborn, A. M. (1990). Deviation from the simple two-parameter model-free approach ti the interpertation of nitrogen-13 nuclear magnetic relaxation of proteins. J. Am. Chem. Soc. 112, 4989-4991.
27. Wittebort, R. J. S., A. (1978). J. Chem. Phys. 69, 1722.
28. Brink, D. M. S., G. R. (1968). Angular Momentum, Clarendom Press, Oxford.
29. Woessner, D. E. (1962). J. Chem. Phys. 37, 647-654.
30. Schurr, J. M., Badcock, H. P. & Fujimoto, B. S. (1994). J. Nucl. Magn. Reson.,Seris B 105.
31. Mandel, A. M., Akke, M. Palmer, A. G. III. (1995). Backbone dynamics of E. Coli ribonuclease HI: correlaions with structure and function in an active enzyme. J. Mol. Biol. 246, 144-163.
32. Henry, E. R. S., A. (1985). J. Chem. Phys. 82, 4753-4761.
33. Dayie, K. T., Wagner, G. & Lefever, L. F. (1996). Theory and paractice of nuclear spin relaxation in proteins. Annu. Rev. Phys. Chem. 47, 243-282.
34. Farrow, N. A., Zhang, O., Szabo, A., Torchia, D. A.,& Kay L. E. (1995). Spectral density function mapping using 15N relaxation data exculsively. J. Biomol. NMR 6, 153-162.
35. Atkinson, R. A. K., B. (2004). The role of protein motions in molecular recognition: insight from heteronuclear NMR relaxation measurements. Progress in Nuclear Magnetic Resonance Spectroscopy 44, 141-187.
36. Leach, A. R. (1996). Molecular modelling: princiles and applications. 2 edit.
37. Verlet, L. (1967). Computer 'Experiments' on Classical Fluids. I. Thermodynamical Properties of Lennard-Jones Molecules. Phys. Rev. 159, 98-103.
38. Berendsen H J C, J. P. M. P., W F van Gunsteren, A Di Nola and J R Haak. (1984). Molecular Dynamics with Coupling to an External Bath. J. Chem.Phys. 81, 3684-3690.
39. Anderson, H. C. (1980). Molecular Dynamics Simulations at Constant Pressure and/or Temperature. J. Chem. Phys. 72, 2384-2393.
40. Ryckaert, J. P., Cicotti,G., and Berensden, H. J. (1977). Numerical Intergration of the Cartesion Equations of Motion of a System with Constraints: Molecular Dynamics of n-Alkanes. J. Compu. Phys. 23, 327-341.
41. Tobias, D. J., and Brooks, C. L. III. (1988). Molecular dynamics with internal coordinate constraints. J. Chem. Phys. 89, 5115-5126.
42. Su, M. S. (1997). Structure and function of E. Coli thioesterase/protease I. Mester Science Thesis.
43. Barbato, G., Ikura, M., Kay L., Pastor, R. & Bax, A. (1992). Backbone dynamics of calmodulin studied by 15N relaxation using inverse detected two-dimensional NMR spectroscopy: the central helix is flexible. Biochemistry 31, 5260-5278.
44. Kay, L. E., Nicholson, L., Delaglio, F., Bax, A. &Torchia, D. (1992). Pulse sequences for remical of the effects of cross correlation between dipolar and chemical-shift anisotropy relaxation mechanisms on the measurement of neteronuclea T1 and T2 values in proteins. J. Nucl. Magn. Reson. 97, 359-375.
45. Piotto, M., Saudek, V. & Sklenar, V. (1992). Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J. Biomol. NMR 2, 661-665.
46. Sklenar, V., Piotto, M., Leppik, R. & Saudek, V. (1993). Gradient-tailored water suppression for 1H-15N HSQC experiments optimized to retain full sensitivity. J. Nucl. Magn. Reson. 102A, 241-245.
47. Markley, J. L., Horsley, W. J. & Klein, M. P. (1971). Spin-lattice relaxation measurements in slowly relaxation complex spectra. J. Chem. Phys. 55, 3604-3605.
48. Wishart, D. S., Bigam, C. G., Yao, J., Abildgaard, F., Dyson, H. J. & Oldfield, E. et al. (1995). 1H, 13C, 15N chemical shift referencing in biomolecular NMR. J. Biomol. NMR 6, 135-140.
49. Wishart, D. S. S., B. D. (1994). Chemical shifts as a tool for structural determination. Methods in Enzymology 239, 363-392.
50. Neidig, K. P., Geyer, M., Goerler, A., Antz, C., Saffrich, R., Beneicke, W. & Kalbitzer, H. R. (1995). AURELIA, a program for computer-aided analysis of multidimentional NMR spectra. J. Biomol. NMR 6, 255-270.
51. Lee, L. K., Rance, M., Chazin, W. J. &Palmer, A. G. III. (1997). Rotational diffusion anisotropy of proteins from simultaneous analysis of 15N and 13C nuclaer spin relaxation. J. Biomol. NMR 9, 287-298.
52. Brueschweiler, R., Liao, X. & Wright, P. E. (1995). Long-range motional restrictions in a multidomain zinc-finger protein anisotropic tumbling. Science 268, 886-889.
53. Kroenke, C. D., Rance, M. & Palmer, A. G. III. (1999). Variability of the 15N chemical shift anisotropy in E. Coli ribonuclease HI in solution. J. Am. Chem. Soc. 121.
54. Palmer, A. G. I. (2001). NMR probes of molecular dynamics: overview and comparision with other techniques. Annu. Rev. Biophys. Biochem. Struct. 30, 125-155.
55. Palmer, A. G. I. C., D. A. (1992). Molecular dynamics analysis of NMR relaxation in a Zinc-finger peptide. J. Am. Chem. Soc. 114, 9059-9067.
56. Huang, Y. T., Liaw, Y. C., Gorbatyuk, V. Y., & Huang, T. H. (2001). Backbone dynamics of E. coli. thioesterase/protease I: Evidence of a fexible active-site enviroment for a serine protease. J. Mol. Biol. 307, 1075-1090.
57. Lin, T. H., Chen, C. P., Huang R. F., Lee, Y. l., Shaw, J. F., & Huang T. H. (1998). Multinuclear NMR resonace assignments and the secondary structure of E. coli thioesterase/protease I: A member of a new subclass of lipolytic enzymes. J. Biomol. NMR 11, 363-380.
58. Tyukhtenko, S. I., Livinchyuk, A.V., Huang, Y.T., Chang, C.F., Shaw, J.F., & Huang, T. H. (2002). NMR Studies of the Hydrogen Bonds Involving the Catalytic Triad of E. coli Thioesterase/Protease I. FEBS Letters 528, 203-206.
59. Volkman, B. F., Lipson, D. Wammer, D.E., & Kern, D. (2001). Two-state allosteric behavior in a single-domain dignaling protein. Science 291, 2429-2433.
60. Upton C., B. J. T. (1995). A new family of lipolytic enzymes? Trend. Biochem. Sci. 20, 178-179.
61. Dalrymple B.P., C. D. H., Layton I., McSweeney C.S., Xue G.-P., Swadling Y.J., Lowry J.B. (1997). Three Neocallimastix patriciarum esterases associated with the degradation of complex polysaccharides are members of a new family of hydrolases. Microbiology 143, 2605-2614.
62. Molgaard A., K. S., Larsen S. (2000). Rhamnogalacturonan acetylesterase elucidates the structure and function of a new family of hydrolases. Structure 8, 373-383.
63. Mulder, F. A. A., Skrynnikov, N. R., Hon, B., Dahlquist, F. W., & Kay, L. E. (2001). Measurement of Slow (s-ms) Time Scale Dynamics in Protein Side Chains by 15N Relaxation Dispersion NMR Spectroscopy: Application to Asn and Gln Residues in a Cavity Mutant of T4 Lysozyme. J. Am. Chem. Soc. 123, 967-975.
64. Akke, M. (2002). NMR methods for characterizing microsecond to millisecond dynamics in recognition and catalysis. Current Opinion of Structural Biology 12, 642-647.
65. McElheny, D., Schnell, J. R.,Lansing, J. C., Dyson, H. J., & Wright, P. E. (2005). Defining the role of active-site loop fluctuations in dihydrofolate reductase catalysis. PNAS 102, 5032-5037.