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研究生:洪慧芝
研究生(外文):Hui-Chih Hung
論文名稱:人類胎盤鹼性磷酯結構安定性及催化機制之研究
論文名稱(外文):Conformational stability and catalytic mechanism of human placental alkaline phosphatase
指導教授:張固剛
指導教授(外文):Gu-Gang Chang
學位類別:博士
校院名稱:國防醫學院
系所名稱:生命科學研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:1999
畢業學年度:87
語文別:中文
論文頁數:202
中文關鍵詞:人類胎盤鹼性磷酯模擬
外文關鍵詞:Huamn placental alkaline phosphatasefoldinghomology modeling
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人類胎盤鹼性磷酯為一具有二個相同雙體之金屬,其活性中心含有二個鋅金屬及一個鎂金屬。大腸桿菌鹼性磷酯之X光繞射立體結構已達2.0 A之解析度,但其它鹼性磷酯之立體結構至今仍未解出。本論文主要為探討人類胎盤鹼性磷酯結構之安定性及金屬在其催化機制上所扮演之角色。我利用化學變性劑,胍溶液(guanidinium chloride)及尿素(urea),來檢查人類胎盤鹼性磷酯結構之安定性。結果在胍溶液引起之變性過程中,會出現一個安定之中間過渡狀態之結構(intermediate),顯示此變性為一兩段式過程(biphasic process)。此兩段式變性過程並非由胍溶液所帶入之鹽所致,而是一個變性-解離同時進行之過程。由結果推知此之四級結構十分安定,完整摺疊(intact folding)之單體並未在胍溶液引起之變性過程中出現。類似之結果亦出現在尿素引起之變性過程,但是此變性為多段式過程(multiphasic denaturation)。此結果顯示人類胎盤鹼性磷酯之次區域(subdomain)結構安定性之不同。此外,此之活性中心結構相當安定,在三級結構消失後,活性才逐漸喪失。
除了結構之安定性外,我亦著墨於鎂離子所引起之活性活化作用,及鋅離子所引起之活性抑制作用。鎂離子所引起之活性活化作用為一緩慢過程,此乃由於分子與鎂離子結合緩慢之故(slow binding activation)。鎂離子能保護免受鋅離子抑制,亦能回復受鋅離子抑制之活性。我試圖以結構的觀點來說明鎂離子及鋅離子所引起活性改變之現象。
人類胎盤鹼性磷酯為一膜蛋白,其活性已在一模擬生物膜系統之逆向微脂粒中進行分析。此之受質,4-硝基酚磷酸,分解所產生之產物為黃顏色之4-硝基酚陰離子;由於4-硝基酚陰離子對逆向微脂粒之中間相(interphase)具親和力,於是產生分配現象(partition),形成無色之4-硝基酚。實驗結果顯示此分配現象與緩衝溶液的種類、界面活性劑之濃度及逆向微脂粒球大小有關。此外,2-胺基-2-甲基丙醇將4-硝基酚從逆向微脂粒之中間相置換至水相之能力,是由於胺基,而非羥基所致。
Human placental alkaline phosphatase is a homodimeric metalloenzyme containing two zinc ions and one magnesium ion. The X-ray structure of E. coli enzyme has been refined to 2.0 ?resolution, however, no tertiary structure of the enzyme from other sources is yet available. In this dissertation, my research is aimed to the study of conformational stability and the role of magnesium ion in the catalytic mechanism of human placental alkaline phosphatase. I investigate the denaturation-renaturation process of the enzyme by its sensitivity to chemical denaturants, guanidinium chloride (GdmCl) or urea. Physical methods, e. g., fluorescence, circular dichroism, and ultracentrifugation, or functional properties, i. e., enzyme activity were used to explore the structure change induced by the denaturants. My results clearly indicate that a stable intermediate state is significantly populated in the GdmCl-induced unfolding process. A clear biphasic unfolding phenomenon was observed. The biphasic phenomenon is not a salt effect and is a simultaneous dissociation-denaturation process. The dimeric structure of th enzyme is thus quite stable, an intact folding monomer does not exist during GdmCl-induced unfolding. Similar chemical stability was also observed in urea denaturation. However, more complex multiple intermediate states detected in urea denaturation indicates the differential stability of subdomains of the enzyme. The enzyme activity was inactivated only after substantial tertiary structure has been changed, suggesting that the active site region is more resistant to chemical denaturant than other structural domains. The urea denaturation of the placental enzyme is also a simultaneous dissociation-denaturation process. Folded monomer never existed in the unfolding process. Complete dissociation occurred only beyond 6 M urea.
Besides protein folding, I am also interested in the kinetic behaviors of the placental enzyme, which is activated by magnesium ion but inhibited by zinc ion. The role of zinc ion in the enzyme molecule is essential for catalysis, however, excess zinc ion inhibited the enzyme activity. Magnesium ion stimulates the enzyme activity to reach a maximal level, which is a slow process. Magnesium ion also protects the enzyme against the inhibition by zinc. I have analyzed the kinetic behavior of the slow activation. I proposed a plausible mechanism to explain the zinc inhibition and magnesium activation from the point of view for the enzyme structure at the active site region.
I have also analyzed the enzyme activity in a biomembranous mimicking reverse micellar system. For the assay of alkaline phosphatase, p-nitrophenyl phosphate is used as the substrate. After hydrolysis, 4-nitrophenolic anions are yellow in alkaline solution. I observed the partitioning of 4-nitrophenol, which is colorless in its non-ionized form, in reverse micellar system. My results clearly indicate that the apparent pKa values of 4-nitrophenol are sensitive to the buffer used and also to the water content of the reverse micellar system. 4-nitrophenol has affinity with the surfactant AOT in carbonate buffer. Binding of 4-nitrophenol with the anionic surfactant polar head hinders ionization resulting in elevation of the pKa value of the phenolic -OH group, which occurs in a gradient manner with the most basic -OH at the interface region. Binding of 4-nitrophenol with AOT was affected by the 2-amino-2-methylpropanol buffer, which perturbates the partition of 4-nitrophenol between the water pool and interface. The perturbation of 4-nitrophenol partition in AOT-reverse micelles in 2-amino-2-methylpropanol buffer is due to the amino group of the buffer molecule, because tert-butylamine, rather than isobutanol, induced the replacement.
COVER
Contents
Content of Figures
Content of Tables
Abbreviations
Chinese Abstract
English Abstract
Introduction
Meterials
Methods
Enzyme Preparation
Enzyme Assay
Enzyme Denaturation
Spectrofluorimetric Analysis
Intrinsic fluorescence experiments
Fluorescence quenching experiments
ANS and bis-ANS binding measurement
Spectropolarimetric Analysis
Molecular Weight Determination by the Sucrose-Density Gradient Ultracentrifugation
Data Analysis for the Denaturation Process
Preparation of AOT-Reverse Micelles in Systems of Various Degrees of Hydration
lonization of 4-Nitrophenol in AOT-Reverse Micelles
Results
Biphasic Denaturation of Human Placental Alkaline Phosphatase in Guanidinium Chloride
Fluorescence spectrum change of human placental alkaline phosphatase after denaturation with guanidinium chloride
Biphasic denaturation of human placental alkaline phosphatase in guanidinium chloride
Circular dichroism spectrum change of human placental alkaline phosphatase after denaturation with guanidinium chloride
Anomalous effects of guanidinium chloride on human placental alkaline phosphatase
Quaternary structure of human placental alkaline phosphatase during guanidinium chloride denaturation
Refolding kinetics of guanidinium chloride denatured human placental alkaline phosphatase
Protection of human placental alkaline phosphatase by phosphate against the guanidinium chloride inactivation
Multiphasic Denaturation of Human Placental Alkaline Phosphatase in Urea
Multiphasic denaturation of human placental alkaline phosphatase induced by urea
Activation and denaturation of human placental alkaline phosphatase by Urea
Exploration of the structural dynamics of human placental alkaline phosphatase during urea denaturation by quenching experiments
Detection of unfolding intermediates during protein denaturation by ANS and bis ANS binding
An unfolding intermediate can be stabilized by guanidinium chloride, but not NaCl, during the urea-induced denaturation of human placental alkaline phosphatase
Quaternary structure of human placental alkaline phosphatase during urea denaturation
Slowing Binding Activation of Magnesium Ion for Human Placental Alkaline Phosphatase
The protection and substitution of magnesium ion for zinc - inhibited human placental alkaline phosphatase
Slow activation by magnesium ion of human placental alkaline phosphatase
Analysis of the structure difference for enzymes with various metal contents by acrylamide quenching
Partition of4-Nitrophenol in the AOT/lsooctane-Reverse Micelles
Partition of 4-nitrophenol in different buffer systems in AOT/isooctane-reverse micelles
Effect of ionic strength or surfactant concentration on the partition of 4-nitrophenol in AOT-reverse micelles
Displacement of 4-nitrophenol from the interface to the water pool region of AOT-reverse micelles
lonization of 4-nitrophenol in AOT-reverse micelles
Discussion
Biphasic Denaturation of Human Placental Alkaline Phosphatase Induced by Guanidinium Chloride
Multiphasic Denaturation of Human Placental Alkaline Phosphatase Induced by Urea
Magnesium Ion is A Slow Binding Activator for Human Placental Alkaline Phosphatase
Partition of 4-Nitrophenol in Aerosol-OT Reverse Micelles
Conclusion
References
Figures
Tables
Appendix
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