Littérature scientifique sur le sujet « Acylphosphatases »

We have demonstrated that acylphosphatase possesses ATP-diphosphohydrolase (apyrase-like) activity. In fact, acylphosphatase first catalyses the hydrolysis of the γ-phosphate group of nucleoside triphosphates, and then attacks the β-phosphate group of the initially produced nucleoside diphosphates, generating nucleoside monophosphates. In constrast, it binds nucleoside monophosphates but does not catalyse their hydrolyses. The calculated kcat values for the nucleoside triphosphatase activity of acylphosphatase are of the same order of magnitude as those displayed by certain G-proteins. An acidic environment enhances the apyrase-like activity of acylphosphatase. The true nucleotide substrates of acylphosphatase are free nucleoside di- and triphosphates, as indicated by the Mg2+ ion inhibition of the activity. We have also demonstrated that, although nucleoside triphosphates are still hydrolysed at pH 7.2 and 37 °C, in the presence of millimolar Mg2+ concentrations this occurs at a lower rate. Taken together with the previously observed strong increase of acylphosphatase levels during induced cell differentiation, our findings suggest that acylphosphatase plays an active role in the differentiation process (as well as in other processes, such as apoptosis) by modulating the ratio between the cellular levels of nucleoside diphosphates and nucleoside triphosphates.

The modulation of expression of the skeletal muscle and erythrocyte acylphosphatase isoenzymes by thyroid hormone has been investigated. Our results indicate a differential regulation of the two enzymic isoforms by tri-iodothyronine (T3) in K562 cells in culture: an increase in the specific mRNA during T3-stimulation is shown only for the skeletal muscle isoenzyme. A fast and transient T3 induction of the accumulation of the specific mRNA can be observed, reaching a maximum 8 h after hormone treatment and then rapidly decreasing almost to the steady-state level after 24 h. A nuclear run-on assay was performed to explore the mechanisms of this regulation. These studies indicate that T3 induction of skeletal muscle acylphosphatase mRNA is due, at least in part, to a fast and transient increase in the rate of gene transcription, within 4 h after hormone administration. A very rapid decrease is then observed within a further 2 h. T3-dependent accumulation of the mRNA for the skeletal muscle acylphosphatase requires ongoing protein synthesis, as confirmed by inhibition with cycloheximide or puromycin. These findings indicate that the transcriptional regulation of the gene may be indirect.

The PhD thesis describes the structural and enzymatic characterization of two new members of the acylphosphatase enzyme family and the state of the art of the phylogenetic distribution of over 500 currently known acylphosphatases. The thesis is also focussed on the amyloid properties of the above mentioned proteins, due to the role that this protein family have played as a model in the study of amyloid aggregation mechanisms.

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

近十年間，離子液體在生物催化和蛋白質化學方面的廣泛應用引起了鑒定蛋白質在離子液體中特性的研究興趣。本研究以古菌Pyrococcus horikoshii的酰基磷酸酶acylphosphatase (PhAcP) 和50% (v/v) 離子液體1-乙基-3-甲基咪唑四氟硼酸鹽 ([EMIM][BF₄]) 作為研究模型，首次利用多維核磁共振譜對蛋白質在離子液體中的結構和穩定性作高解析度的分析。我們首先通過蛋白質主鏈共振歸屬，得出PhAcP每個被歸屬的胺基酸在50% (v/v) [EMIM][BF₄] 中¹³C[superscript α]、¹³C[superscript β]、¹³CO、¹⁵N、H[superscript N]和H[superscript α]原子的化學位移。¹³C的化學位移相對無序纏捲狀態的¹³C化學位移的偏差分析 (¹³C secondary shift)，以及二級結構之間的nuclear Overhauser effect (NOE) 連接顯示PhAcP在50% (v/v) [EMIM][BF₄] 中的二級結構與PhAcP的自然結構大致相同，其三級結構亦無顯著變化。此外，我們以二維的¹H-¹⁵N HSQC實驗觀察在318K、328K和338K這三個溫度下的硫氰酸胍 (GdnSCN) 誘導蛋白質變性，發現同一溫度下無論50% (v/v) [EMIM][BF₄]是否存在，PhAcP的變性曲線都互相重疊，得到的 [GdnSCN]₁[subscript /]₂值也相同，由此可推斷50% (v/v) [EMIM][BF₄] 對PhAcP的穩定性沒有影響。
The extensive application of ionic liquid in biocatalysis and protein chemistry in the past decade arouses interest in the characterization of protein behavior in ionic liquid. This study demonstrates the use of multi-dimensional nuclear magnetic resonance (NMR) spectroscopy to investigate the structure and conformational stability of protein in ionic liquid at a high resolution for the first time, with Pyrococcus horikoshii acylphosphatase (PhAcP) and 50% (v/v) 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIM][BF₄]) as a study model. The backbone amide resonances of PhAcP in 50% (v/v) [EMIM][BF₄] were assigned in order to obtain the chemical shifts of ¹³C[superscript α], ¹³C[superscript β], ¹³CO, ¹⁵N, HN and H[superscript α] of each assigned residue. The estimation of secondary structure by the ¹³C secondary shift analysis and the nuclear Overhauser effect (NOE) connectivities observed within secondary structures together suggest that PhAcP has secondary structures arranged in native-like topology and there is no major alteration in the tertiary structure in 50% (v/v) [EMIM][BF₄]. Guanidine thiocyanate (GdnSCN)-induced denaturation was performed at 318K, 328K and 338K and monitored by 2D ¹H-¹⁵N HSQC experiments to study the conformational stability of PhAcP in 50% (v/v) [EMIM][BF₄]. The overlapping denaturation curves and consistent [GdnSCN]₁[subscript /]₂ values obtained at each temperature indicate no observable trend of stability alteration.
Detailed summary in vernacular field only.
Lee, Tsz Ying.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2013.
Includes bibliographical references (leaves 57-63).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts also in Chinese.
Abstract --- p.i
摘要 --- p.ii
Acknowledgements --- p.iii
Contents --- p.iv
Abbreviations --- p.vii
List of Figures --- p.viii
List of Tables --- p.ix
Chapter Chapter 1 --- Introduction --- p.1
Chapter 1.1 --- Introduction to ionic liquid --- p.1
Chapter 1.1.1 --- Ionic liquid as reaction medium, co-solvent and additive in biocatalysis and protein chemistry --- p.1
Chapter 1.1.2 --- The impact of ionic liquid on protein structure and stability is poorly understood --- p.3
Chapter 1.2 --- PhAcP in [EMIM][BF₄] as a model to study the structure and stability of protein in ionic liquid by NMR spectroscopy --- p.6
Chapter 1.2.1 --- The application of [EMIM][BF₄] with protein --- p.6
Chapter 1.2.2 --- The background of PhAcP --- p.9
Chapter 1.2.3 --- Overview of the study --- p.10
Chapter Chapter 2 --- Materials and Methods --- p.12
Chapter 2.1 --- Expression and purification of PhAcP --- p.12
Chapter 2.1.1 --- Expression of PhAcP in Escherichia coli system --- p.12
Chapter 2.1.2 --- Purification of PhAcP --- p.14
Chapter 2.2 --- Solubility determination --- p.15
Chapter 2.3 --- NMR experiments --- p.17
Chapter 2.3.1 --- General procedures and sample preparation --- p.17
Chapter 2.3.2 --- ¹H-¹⁵N HSQC spectra in various concentrations of [EMIM][BF₄] --- p.18
Chapter 2.3.3 --- Structural characterization --- p.18
Chapter 2.3.4 --- Stability characterization --- p.19
Chapter Chapter 3 --- Results --- p.21
Chapter 3.1 --- Can the solubility of PhAcP in [EMIM][BF₄] reach the millimolar range required for NMR study? --- p.21
Chapter 3.2 --- Determination of the [EMIM][BF₄] concentration for a feasible NMR study --- p.23
Chapter 3.3 --- Backbone resonance assignment of PhAcP in 50% (v/v) [EMIM][BF₄] --- p.26
Chapter 3.4 --- Structural characterization of PhAcP in 50% (v/v) [EMIM][BF₄] --- p.29
Chapter 3.4.1 --- Secondary structure estimation by ¹³C secondary shifts --- p.29
Chapter 3.4.2 --- NOE connectivities within secondary structures --- p.35
Chapter 3.5 --- Characterization of the conformational stability of PhAcP in 50% (v/v) [EMIM][BF₄] by guanidine thiocyanate-induced denaturation --- p.40
Chapter Chapter 4 --- Discussion --- p.46
Chapter 4.1 --- The structure of PhAcP in 50% (v/v) [EMIM][BF₄] resembles the native conformation --- p.46
Chapter 4.2 --- The conformational stability of PhAcP has no observable change in 50% (v/v) [EMIM][BF₄] --- p.47
Chapter 4.3 --- Insight into the application of enzyme in ionic liquid --- p.48
Chapter 4.4 --- Limitation of the study --- p.49
Chapter 4.5 --- Insight into future studies --- p.50
Chapter Chapter 5 --- Conclusions --- p.51
Appendix --- p.53
References --- p.57