Crystal structure of human common-type acylphosphatase and insights into enzyme-substrate interaction.

• Other Authors: Yeung, Ching Yee.
• Format: Others
• Language: English; Chinese
• Published: 2008
• Subjects: Hydrolases; Acid Anhydride Hydrolases
• Online Access: http://library.cuhk.edu.hk/record=b5893591; http://repository.lib.cuhk.edu.hk/en/item/cuhk-326563

Yeung, Ching Yee. === Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. === Includes bibliographical references (leaves 112-122). === Abstracts in English and Chinese. === Acknowledgments --- p.I === Abstract --- p.II === 摘要 --- p.III === Content --- p.IV === Abbreviations and symbols --- p.XI === List of tables and figures --- p.XV === Chapter Chapter 1 --- Introduction --- p.1 === Chapter 1.1 --- Acylphosphatase --- p.1 === Chapter 1.2 --- Human acylphosphatase --- p.4 === Chapter 1.3 --- Hyperthermophilic Pyrococcus horikoshii acylphosphatase --- p.5 === Chapter 1.4 --- Human common-type acylphosphatase as a mesophilic homologue of Pyrococcus horikoshii acylphosphatase --- p.8 === Chapter 1.5 --- Enzyme-substrate interaction of acylphosphatase --- p.9 === Chapter Chapter 2 --- Materials and methods --- p.10 === Chapter 2.1 --- Preparation of Escherichia coli competent cells --- p.10 === Chapter 2.2 --- SDS-polyacrylamide gel electrophoresis --- p.11 === Chapter 2.2.1 --- Preparation of polyacrylamide gel --- p.11 === Chapter 2.2.2 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.12 === Chapter 2.2.3 --- Staining of protein in polyacrylamide gel by Coommassie Brillant Blue R250 --- p.12 === Chapter 2.3 --- Expression and purification of Protein --- p.13 === Chapter 2.3.1 --- "General bacterial culture, harvesting and lysis" --- p.13 === Chapter 2.3.2 --- Purification of acylphosphatase --- p.14 === Chapter 2.3.2.1 --- Ion-exchange chromatography --- p.14 === Chapter 2.3.2.2 --- Size excision chromatography --- p.15 === Chapter 2.3.3 --- Protein concentration determination --- p.16 === Chapter 2.4 --- X-ray crystallography --- p.17 === Chapter 2.4.1 --- Crystallization of Hu CT AcP --- p.17 === Chapter 2.4.2 --- Model building and structural refinement --- p.18 === Chapter 2.4.3 --- Crystallization of Hu CT AcP -substate analogue complex --- p.19 === Chapter 2.5 --- Enzymatic Assay --- p.21 === Chapter 2.5.1 --- Preparation of benzoyl phosphate --- p.21 === Chapter 2.5.2 --- Purity check of the BP synthesized --- p.22 === Chapter 2.5.3 --- Determination of kinetic parameters of Hu CT AcP --- p.25 === Chapter 2.5.4 --- Determination of Ki value of substrate analogue --- p.27 === Chapter 2.6 --- Isothermal titration calorimetry --- p.28 === Chapter 2.7 --- Reagents and Buffers --- p.30 === Chapter 2.7.1 --- Reagent for competent cell preparation --- p.30 === Chapter 2.7.2 --- Media for bacterial culture --- p.31 === Chapter 2.7.3 --- Reagent for SDS-PAGE --- p.32 === Chapter 2.7.4 --- Buffer for AcP purification --- p.33 === Chapter 2.7.5 --- Buffer for enzymatic assay and ITC --- p.33 === Chapter Chapter 3 --- Structural determination of human common-type acylphosphatase --- p.34 === Chapter 3.1 --- Introduction --- p.34 === Chapter 3.2 --- Expression and purification of Hu CT AcP --- p.35 === Chapter 3.3 --- Structure of Hu CT AcP was determined by X-ray crystallography --- p.37 === Chapter 3.3.1 --- Crystallization of Hu CT AcP --- p.37 === Chapter 3.3.2 --- Model building and structural refinement --- p.41 === Chapter 3.3.3 --- Hu CT AcP shares a same α/β sandwich fold structure as other AcP --- p.43 === Chapter 3.4 --- Discussion --- p.46 === Chapter 3.4.1 --- Active site structure of Hu CT AcP is the same as those of bovine CT AcP and Ph AcP --- p.46 === Chapter 3.4.2 --- Absence of salt bridge between the active site residue and the C-terminal may contribute to the higher catalytic efficiency of Hu CT AcP --- p.52 === Chapter Chapter 4 --- Characterization of interaction between acylphosphatase and substrate analogues --- p.56 === Chapter 4.1 --- Introduction --- p.56 === Chapter 4.2 --- Selected substrate analogues --- p.57 === Chapter 4.3 --- Characterization of AcP-substrate analogue interaction by enzymatic assay --- p.59 === Chapter 4.3.1 --- Enzyme kinetics of Hu CT AcP was determined by the continuous optical assay of BP hydrolysis --- p.59 === Chapter 4.3.2 --- Substrate analogues were found to be competitive inhibitor to the AcP-catalyzed BP hydrolysis --- p.61 === Chapter 4.3.3 --- S-BA was the best competitive inhibitor against AcP-catalyzed BP hydrolysis --- p.64 === Chapter 4.3.4 --- S-BA was shown to be a competitive inhibitor for both Hu CT and Ph AcP --- p.66 === Chapter 4.4 --- Characterization of AcP-substrate analogue interaction by thermodynamic study --- p.68 === Chapter 4.4.1 --- Enthalpy change was observed for the association between substrate analogue and AcP --- p.68 === Chapter 4.4.2 --- S-BA was shown to bind Hu CT AcP with high affinity in ITC study --- p.68 === Chapter 4.5 --- S-BA was found to be the best substrate analogue for AcP --- p.72 === Chapter 4.6 --- Discussion --- p.73 === Chapter 4.6.1 --- Structure-affinity study of substrate analogue reveals chemical structures essential to interaction with AcP --- p.73 === Chapter 4.6.2 --- Structure-affinity study of substrate analogues is consistent with docking model of AcP with acetyl phosphate --- p.75 === Chapter 4.6.3 --- Validation of docking model by crystal complex structure --- p.78 === Chapter 4.6.4 --- Structural basis of substrate inhibition in Hu CT AcP --- p.80 === Chapter 4.6.4.1 --- Substrate inhibition is observed in Hu CT AcP --- p.80 === Chapter 4.6.4.2 --- Non-productive binding and substrate inhibition in AcP --- p.80 === Chapter Chapter 5 --- Investigation on the effect of salt bridge on acylphosphatase- substrate analogue interaction --- p.84 === Chapter 5.1 --- Introduction --- p.84 === Chapter 5.2 --- Thermodynamic study on the binding of S-BA with AcPs --- p.87 === Chapter 5.2.1 --- Determination of thermodynamic parameters of interaction between AcP and substrate analogue --- p.87 === Chapter 5.2.2 --- Determination of thermodynamic parameters as a function of temperature --- p.90 === Chapter 5.3 --- Discussion --- p.93 === Chapter 5.3.1 --- The presence of salt bridge leads to a reduced flexibility at the substrate binding active site --- p.93 === Chapter 5.3.2 --- The single salt bridge reduces the flexibility of active site in both study on thermodynamics of binding and thermodynamics of activation --- p.94 === Chapter 5.3.3 --- Temperature dependence of the thermodynamic parameters and heat capacity change ΔCp --- p.97 === Chapter 5.3.3.1 --- Change in heat capacity reveals the nature of the complex interface --- p.97 === Chapter 5.3.3.2 --- Determination of heat capacity change ΔCp --- p.98 === Chapter Chapter 6 --- Structural determination of acylphosphatase-substrate analogue complex --- p.102 === Chapter 6.1 --- Introduction --- p.102 === Chapter 6.2 --- Soaking and cocrystallization failed to give cocrystal structure of Hu CT AcP and S-BA --- p.103 === Chapter 6.4 --- Discussion --- p.106 === Chapter 6.4.1 --- Hu CT AcP and S-BA is not compatible with cocrystal formation --- p.106 === Chapter 6.5 --- Future prospect --- p.107 === Chapter 6.5.1 --- Structure determination by NMR spectroscopy --- p.107 === Chapter 6.5.2 --- Structure determination of AcP with aluminofluoride complexes --- p.108 === Chapter Chapter 7 --- Conclusion --- p.109 === Reference --- p.112