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

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2009. / Vita. Includes bibliographical references.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21781号 / 工博第4598号 / 新制||工||1716(附属図書館) / 京都大学大学院工学研究科物質エネルギー化学専攻 / (主査)教授 辻 康之, 教授 大江 浩一, 教授 近藤 輝幸 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

<p>In the present thesis the density functional B3LYP has been used to study reactions of NO and O₂ in redox active enzymes.</p><p>Reduction of nitric oxide (NO) to nitrous oxide (N₂O) is an important part in the bacterial energy conservation (denitrification). The reduction of NO in three different bimetallic active sites leads to the formation of hyponitrous acid anhydride (N₂O₂^2-). The stability of this intermediate is crucial for the reaction rate. In the two diiron systems, respiratory and scavenging types of NOR, it is possible to cleave the N-O bond, forming N₂O, without any extra protons or electrons. In a heme-copper oxidase, on the other hand, both a proton and an electron are needed to form N₂O.</p><p>In addition to being an intermediate in the denitrification, NO is a toxic agent. Myoglobin in the oxy-form reacts with NO forming nitrate (NO₃ ^-) at a high rate, which should make this enzyme an efficient NO scavenger. Peroxynitrite (ONOO^-) is formed as a short-lived intermediate and isomerizes to nitrate through a radical reaction.</p><p>In the mechanism for pumping protons in cytochrome oxidase, thermodynamics, rather than structural changes, might guide protons to the heme propionate for further translocation.</p><p>The dioxygenation of arachidonic acid in prostaglandin endoperoxide H synthase forms the bicyclic prostaglandin G₂, through a cascade of radical reactions. The mechanism proposed by Hamberg and Samuelsson is energetically feasible.</p>

In the present thesis the density functional B3LYP has been used to study reactions of NO and O2 in redox active enzymes. Reduction of nitric oxide (NO) to nitrous oxide (N2O) is an important part in the bacterial energy conservation (denitrification). The reduction of NO in three different bimetallic active sites leads to the formation of hyponitrous acid anhydride (N2O22-). The stability of this intermediate is crucial for the reaction rate. In the two diiron systems, respiratory and scavenging types of NOR, it is possible to cleave the N-O bond, forming N2O, without any extra protons or electrons. In a heme-copper oxidase, on the other hand, both a proton and an electron are needed to form N2O. In addition to being an intermediate in the denitrification, NO is a toxic agent. Myoglobin in the oxy-form reacts with NO forming nitrate (NO3 -) at a high rate, which should make this enzyme an efficient NO scavenger. Peroxynitrite (ONOO-) is formed as a short-lived intermediate and isomerizes to nitrate through a radical reaction. In the mechanism for pumping protons in cytochrome oxidase, thermodynamics, rather than structural changes, might guide protons to the heme propionate for further translocation. The dioxygenation of arachidonic acid in prostaglandin endoperoxide H synthase forms the bicyclic prostaglandin G2, through a cascade of radical reactions. The mechanism proposed by Hamberg and Samuelsson is energetically feasible.

<p>This thesis presents methods for air sampling and determination of isocyanates, amines, aminoisocyanates and anhydrides. These organic compounds are generated during thermal degradation of polymers such as polyurethane (PUR) or epoxy.</p><p>Isocyanates, amines and anhydrides are airway irritants known to cause occupational asthma. Some of the compounds are listed as human carcinogens. Many workers are exposed.</p><p>Isocyanates and anhydrides are reactive and needs to be immediately derivatized during sampling. Methods have been developed for determination of airborne isocyanates, aminoisocyanates and anhydrides using di-n-butylamine (DBA) as reagent to form stabile urea derivatives or amide derivatives. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) enabled detection limits as low as 10 attomoles. A nitrogen-selective LC-detector enabled quantification of DBA-derivatives in reference solutions. </p><p>A novel sampler is presented. The sampler consists of a denuder in series with a three-stage cascade impactor and an end filter. The sampler made it possible to reveal the distribution of isocyanates between gas and different particle size fractions. During thermal degradation of PUR, isocyanates were associated to particle size fractions (<1 µm) that may penetrate to the lower airways. The distribution during 8 minutes changes noticeably. Aromatic isocyanates become associated to small particles (<1 µm). As a reference method, air-sampling was performed using an impinger filled with di-n-butylamine (DBA) in toluene, connected in series with a glass fiber filter. There was a good agreement between the denuder-impactor sampler and the reference method.</p>

This thesis presents methods for air sampling and determination of isocyanates, amines, aminoisocyanates and anhydrides. These organic compounds are generated during thermal degradation of polymers such as polyurethane (PUR) or epoxy. Isocyanates, amines and anhydrides are airway irritants known to cause occupational asthma. Some of the compounds are listed as human carcinogens. Many workers are exposed. Isocyanates and anhydrides are reactive and needs to be immediately derivatized during sampling. Methods have been developed for determination of airborne isocyanates, aminoisocyanates and anhydrides using di-n-butylamine (DBA) as reagent to form stabile urea derivatives or amide derivatives. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) enabled detection limits as low as 10 attomoles. A nitrogen-selective LC-detector enabled quantification of DBA-derivatives in reference solutions. A novel sampler is presented. The sampler consists of a denuder in series with a three-stage cascade impactor and an end filter. The sampler made it possible to reveal the distribution of isocyanates between gas and different particle size fractions. During thermal degradation of PUR, isocyanates were associated to particle size fractions (&lt;1 µm) that may penetrate to the lower airways. The distribution during 8 minutes changes noticeably. Aromatic isocyanates become associated to small particles (&lt;1 µm). As a reference method, air-sampling was performed using an impinger filled with di-n-butylamine (DBA) in toluene, connected in series with a glass fiber filter. There was a good agreement between the denuder-impactor sampler and the reference method.