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研究生:陳淑婷
研究生(外文):Shu-Ting Chen
論文名稱:人類六磷酸葡萄糖異構酵素之遺傳突變點對活性及穩定性的影響
論文名稱(外文):The Effects of Hereditary Mutations of Phosphoglucose Isomerase on Its Catalytic Efficiency and Structural Stability
指導教授:孟孟孝
指導教授(外文):Menghsiao Meng
學位類別:碩士
校院名稱:國立中興大學
系所名稱:農業生物科技學研究所
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:83
中文關鍵詞:六磷酸葡萄糖異構酵素遺傳突變點穩定性
外文關鍵詞:Phosphoglucose IsomeraseStructural StabilityHereditary Mutations
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  • 被引用被引用:4
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6-磷酸葡萄糖異構酵素(phosphoglucose isomerase,E.C. 5.3.1.9;簡稱PGI),在生理上主要參與醣類代謝中的醣解作用(glycolysis)及醣質新生作用(gluconeogenesis)中6-磷酸葡萄糖(6-phosphoglucose,G6P)與6-磷酸果糖(6-phosphofructose,F6P)間的轉換,由於6-磷酸葡萄糖和6-磷酸果糖在醣解作用、醣質新生作用及五碳醣磷酸循環(pentose phosphate cycle)扮演極為重要的角色,因此當人體6-磷酸葡萄糖異構酵素因突變而發生缺損時,會造成遺傳性溶血性貧血(Hereditary nonspherocytic haemolytic anaemia)。為了探討先天遺傳突變對6-磷酸葡萄糖異構酵素結構、活性及穩定性的影響,我們將人體6-磷酸葡萄糖異構酵素的基因選殖入pUC18,並利用點突變法(Site-direct mutagenesis)構築具有先天性突變的6-磷酸葡萄糖異構酵素基因,然後將先天性突變的6-磷酸葡萄糖異構酵素基因送入大腸桿菌表現,再利用陰離子交換層析管柱及陽離子交換層析管柱純化蛋白,最後進行酵素活性及穩定性的分析。各突變酵素對受質F6P的酵素催化反應速率常數(kcat/KM)分別如下:R83W(Arg83突變為 Trp之變異酵素,以下之突變以此原則命名)、V101M、R347C及R472H約為wild type(WT)的50 ﹪,R347H 約為WT的30 ﹪,而I524T約為WT的5 ﹪。另外R273H即使在分析時加入大量酵素下也無法測得活性,而S278L及E495K則無法以同樣純化方法獲得蛋白,而且在生產此兩蛋白之細胞萃取液中也無法測得活性。在熱穩定方面,我除了測量各個突變酵素在50℃的熱穩定性外,還測量各個突變酵素在過量的F6P(1 mM)存在下及正常生理濃度的F6P (10 μM)下的半衰期(t1/2)。結果顯示除了I524T的熱穩定性與WT差異不大外,其餘的突變蛋白即使在加入1 mM 的F6P下,熱穩定性也遠低於WT。配合已知的人類6-磷酸葡萄糖異構酵素之立體結構,本篇論文也嘗試討論各個突變點影響蛋白穩定性的原因。未來希望繼續完成其他突變蛋白的分析。在各個突變點造成結構上的推測,我也想用更多的實驗證據來證明我的論點。
. Phosphoglucose isomerase (E C 5.3.1.9;PGI) is a dimeric enzyme that catalyzes the reversible inter-conversion of glucose-6-phosphate(G-6-P)and fructose-6-phosphate(F-6-P) in glycolysis and gluconeogenesis pathways. Phosphoglucose isomerase deficiency is the third most common enzyme deficiency known to cause hereditary nonspherocytic hemolytic anemia. Our aim is to analyze the catalytic efficiency and structural stability of the inherited mutant phosphoglucose isomerase. The mutated proteins were generated by site-directed mutagenesis and expressed in E. coli. The activity assays show that mutant proteins R83W、V101M、R347C and R472H decrease approximately 2 folds, R347H decreases approximately 3 folds, and I524T decreases approximately 20-fold compare with the kcat/KM of wide type enzyme. R273H has no activity even a large amount of the enzyme was used for activity assay. S278L and E495K could not be obtained under the same purification conditions suggesting that they have problem in protein folding. Regarding thermostability, the half-lives (t1/2) of mutated proteins were much less stable than WT event at saturated concentration of substrate. I524T is slightly unstable compared with wild-type enzyme at 55 ℃ with 1 mM F-6-P. Combined with the 3-D structure of human phosphoglucose isomerase, I also attempt to address the reason causing the decrease of thermostability of each mutant PGI protein. More hereditary mutated PGI proteins will be characterized in the future to address the structure-function relationship of the hereditary mutations of phosphoglucose isomerase.
目錄 頁
中文摘要 I
英文摘要 III
目錄 IV
圖目錄 VI
表目錄 IX
壹、 前言 1
6-磷酸葡萄糖異構酵素之功能 1
6-磷酸葡萄糖異構酵素之X光結晶學研究 4
目前對6-磷酸葡萄糖異構酵素提出的催化機制 8
研究動機與目的 11
貳、 材料與方法 14
質體之構築介紹 14
菌株、質體及培養基 14
聚合酶鏈鎖反應定點突變法 14
DNA 黏接反應 16
質體轉型作用 16
核苷酸定序法鑑定突變株 17
酵素大量製備及純化 18
蛋白質電泳分析 20
蛋白質定量 21
6-磷酸葡萄糖異構酵素之氨基酸定序比對分析 22
酮醣定性分析 23
酵素動力學分析 25
參、結果 30
人類6-磷酸葡萄糖異構酵素基因的蛋白表現 30
人類6-磷酸葡萄糖異構酵素的純化與比對 31
催化6-磷酸果醣轉換成6-磷酸葡萄糖的酵素動力學分析 32
催化6-磷酸葡萄糖轉換成6-磷酸果醣的酵素動力學分析 33
6-磷酸葡萄糖異構酵素的熱穩定分析 34
肆、討論 35
伍、未來展望 41
陸、圖和表 42
柒、參考文獻                      74
Achari A, Marshall SE, Muirhead H, Palmieri RH, and Noltmann EA 1981. Glucose-6-phosphate isomerase Phil. Trans. R. Soc. Lond. B 293:145-
Andersson SG, Zomorodipour A, Andersson JO, Sicheritz-Ponten T, Alsmark UC, Podowski RM, Naslund AK, Eriksson AS, Winkler HH, Kurland CG. 1998. The genome sequence of Rickettsia prowazekii and the origin of mitochondria. Nature 396:133-
Baronciani L, Zanella A, Bianchi P, Zappa M, Aalfinito F, Lolascon A, Tannoia N, Beutler E, Sirchia. 1996. Study of the molecular defects in glucosephosphate isomerase deficient patients affected by chronic hemolytic anamia. Blood 88:2306-.
Baumann M, Kappl A, Lang T, Brand K, Siegfried W, Paterok E. 1990. The diagnostic validity of the serum tumor marker phosphohexose isomerase (PHI) in patients with gastrointestinal, kidney, and breast cancer. Cancer Invest 8:351-
Baughan MA, Valentine WN, Paglia DE, Ways PO, Simson ER , DeMarsh QB . 1968 . Hereditary hemolytic anemia associated with glucose phosphate isomerase (GPI) deficiency. A new enzyme defect. of human erythozytes. Blood 32:236-
Baumann M. and Brand K. 1988. Purification and characterization of phosphohexose isomerase from human gastrointestinal carcinoma and its potential relationship to neuroleukin. Cancer Res 48:7018-.
Baumgart E, Fahimi HD, Stich A, Volkl A. 1996 L-lactate dehydrogenase A4- and A3B isoforms are bona fide peroxisomal enzymes in rat liver. Evidence for involvement in intraperoxisomal NADH reoxidation. J Biol Chem 271:3846-
Beutler E, West C, Britton HA, Harris J, Forman L. 1997 Glucosephosphate isomerase(GPI)deficiency mutations associated with hereditary hemolytic anemia(HNSHA). Blood Cellls Mol. Dis 23:402-
Cao MJ, Osatomi K, Matsuda R, Ohkubo M, Hara K, Ishihara T. 2000 Purification of a novel serine proteinase inhibitor from the skeletalmuscle of white croaker. Biochem Biophys Res Commun 272:485-
CCowie DB, Cohen GN. 1957. Biosynthesis by Escherichia coli of active altered proteins containing selenium instead of sulfur. Biochim Biophys Acta 26:252-
Chaput M, Claes V, Portetelle, Cludts I, Cravador A, Burny A, Gras H, and Tartar, A. 1988. The neurotrophic factor neuroleukin is 90% homologus with phosphoglucose isomerase. Nature 332:454-
Chatterjee G. V. , Noltmann EA 1967. Dye-sensitized photooxidation as a tool for the elucidation of critical amino acid residues in phosphoglucose isomerase. Eur J Biochem 2: 9-
Chiao JW, Andreeff M, Freitag WB, Arlin Z. 1982. Induction of in vitro proliferation and maturation of human aneuploid myelogenous leukemic cells. J Exp Med 155:1357-
Chiao JW, Freitag WF, Steinmetz JC, Andreeff M. 1981. Changes of cellular markers during differentiation of HL-60 promyelocytes to macrophages as induced by T lymphocyte conditioned medium. Leuk Res 5:477-
Chirgwin JM, Noltmann EA. 1975. The enediolate analogue 5-phosphoarabinonate as a mechanistic probe for phosphoglucose isomerase. J Biol Chem 250:7272-
Chou CC, Sun YJ, Meng M, Hsiao CD. 2000 The crystal structure of phosphoglucose isomerase/autocrine motility factor/neuroleukin complexed with its carbohydrate phosphate inhibitors suggests its substrate/receptor recognition. J Biol Chem 275:23154-
Cini, J.K. and Gracy, R.W. 1986 Molecular basis of the isozymes of bovine glucose-6-phosphate isomerase. Arch Biochem Biophys 249:500-
Dische Z, Borenfreund E. 1951. A new spectrophotometric method for the detection and determination of keto sugars and trioses. J Biol Chem 266: 583-.
Dyson JED, Noltmann EA. 1968. The effect of pH and temperature on the kinetic parameters of phosphoglucose isomerase. J Biol Chem 243: 1401-.
Ernest Beutler1, CarolWest1, Howard A. Britton2, John Harris3, Linda Forman1. 1997. Glucosephosphate Isomerase (GPI) Deficiency Mutations Associated with Hereditary Nonspherocytic Hemolytic Anemia (HNSHA) . Blood Cells, Molecules, and Diseases 31: 402—
Faik P, Walker JIH, Redmill AAM, and Morgan MJ. 1988. Mouse glucose-6-phosphate isomerase and neuroleukin have idential 3’ sequences. Nature. 332: 455-
Fersht AR, Shi J, Jack K, Lowe DM, Wilkinson AJ, Blow DM, Brick P, Carter P, Waye MMY, Winter G. 1985. Hydrogen bonding and biological specificity analysed by protein engineering. Nature 314: 235-
Fujii H, Kanno H, Hirono A, Miwa S. 1996. Hematologicallyimportant mutations: Molecular abnormalities of glucosephosphate isomerase deficiency. Blood Cells MolDis 22: 96-
Gibson DR, Gracy RW. 1980. Affinity labeling and characterization of the active site histidine of glucosephosphate isomerase sequence homology with triosephosphate isomerase. J Biol Chem 255: 9369-
Gracy RW, Noltmann EA.1968. Studies on phosphomannose isomerase. II characteristic as a znic metalloenzyme. J Biol chem 243: 4109-
Green JBA, Wright APH, Cheung WY, Lancashire, WE, Hartley BS. 1988. The structure and regulation of phosphoglucose isomerase in Saccharomyces cerevisiae. Mol Gen Genet 215: 100-
Gurney ME, Apatoff BR, Spear GT, Baumel MJ, Antel JP, Bania MB, Reder AT. 1986 Neuroleukin: a lymphokine product of lectin-stimulated T cells. Science 1986 234:574-
Hemsley A, Arnhrim N, Toney MD, Cortopassi G, Galas DJA. 1989. Simple method for site directed mutagenesis using the polymerase chain reaction. Nucleic acids Res. 16:6545-.
Herrera LS, Pascual C. 1978. Genetical and biochemical studies of glucosephosphate isomerase deficient mutants in Saccharomyces cerevisiae. J Gene Micobiology 108:305.-
Hisaichi Fujii, Hitoshi Kanno, KIRA Hirono, Shiro Miwa. 1996. Hematologically important mutation: molecular abnormalities of glucose phosphate isomerase deficiency. Blood Cells Mol Dis 15:96-
Hitoshi K, Hisaichi F, Akira H, Yoji I, Shoichi O, Yasuhiko F, Kenji M, Satoru O, Shiro M. 1996. Molecular analysis of glucose phosphate deficiency associate with hereditary hemolytic anemia . Blood 15:2321-
Hsiao DH, Chou CC, Hsiao YY, Sun YJ, Meng MH. 1997. Expression, purification, and crystallization of two isozymes of 6-phosphoglucose isomerase of Bacillus stearothermophilus, J. Struc. Biol 120:196-
Huppke P, Eunsch D, Pekrun A, Kind R, Winkler H, Schroter W, Lakomek M. 1997. Glucose phosphate isomerase deficiency: biochemical and molecular genetic studies on the enzyme varants of two patients with severe haemolytic anamia, Eur. J Pedatl 156:605-
James GT, Noltmann EA. 1973. Chemical studies on the subunit structure of rabbit muscle phosphoglucose isomerase. J Biol Chem 248:730-
Jeffery CJ, Bahnson BJ, Chien W, Ringe D, Petsko GA. 2000.Crystal structure of rabbit phosphoglucose isomerase, a glycolytic enzyme that moonlights as neuroleukin, autocrine motility factor, and differentiation mediator. Biochemistry 39:955-
Jeffery CJ. Moonlighting proteins. 1999 Trends Biochem Sci 24:8-
Kanno H, FujiiH, Miwa S. 1998.Expression and enzymatic characterization of human glucosephosphate isomerase(GPI)variants accounting for GPI deficiency. 24:54-
Jon Read, Jake Pearce, Xiaochun Li, Hilary Muirhead John Chirgwin and Christopher Davies. 2001. The Crystal Structure of Human Phosphoglucose Isomerase at 1.6 A Resolution: Implications forCatalytic Mechanism, Cytokine Activity and Haemolytic AnaemiaJ. Mol. Biol. 309; 447-
Kemp TD, Nakagawa Y, Noltmann EA. 1974.Physical and chemical properties of yeast phosphoglucose isomerase. J Biol Chem 249:4617-
Liotta LA, Mandler R, Murano G, Katz DA, Gordon RK, Chiang PK, Schiffmann E. 1986 Tumor cell autocrine motility factor. Proc Natl Acad Sci U S A 83:3302-
Lohmann K. 1933. Uber phosphorylierung und Dephosphorylierung. Bildung der naturlichen Hexosemonophosphorsaure aus ihren Komponenten. Biochem.Z. 262,137
Lu HS, Talent JM, Gracy RW. 1981. Chemical modification of critical residues of lysine, arginine, and tryptophan in human phosphoglucose isomerase. J Biol Chem 256:785-
Malaisse WJ, Malaisse-Lagae F, Liemans V, Ottinger R, Willem R., 1990. Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates: isotopic discrimination between hydrogen and deuterium. Mol Cell Biochem 93:153-
Matsumoto I, Staub A, Benoist C, Mathis D. 1999. Arthritis provoked by linked T and B cell recognition of a glycolytic enzyme. Science 286:1732-
Meng M, Chane TL, Sun YJ, Hsiao CD. 1999. Probing the location and function of the conserved histidine residueof phosphoglucose isomerase by using an active site directedinhibitor N-bromoacetylethanolamine phosphate. Protein Science 8:2438-
Meng M, Chen YT, Hsiao YY, Itoh Y, Bagdasarian M. 1998. Mutational analysis of the conserved cationic residues of Bacillus stearothermophilus 6-phosphoglucose isomerase. Eur J Biochem 257:500-
Mizrachi Y. 1989. Neurotrophic activity of monomeric glucophosphoisomerase was blocked by human immunodeficiency virus (HIV-1) and peptides from HIV-1 envelope glycoprotein. J Neurosci Res 23:217-
Monica S, Dennis R, Burton, Henrik J, Ditzel . 2001. Autoantibodies to GPI in rheumatoid arthritis : Linkage between an animal model and human disease. Nature immunology 2:746-
Muirhead H, Shaw PJ. 1974. Three-dimensional structure of pig muscle phosphoglucose isomerase at 6Å resolution. J Mol Biol 89:195-
Muirhead H, Shaw PJ. 1977. Crystallographic structure analysis of glucose-6-phosphate at 3.5Å resolution. J Mol Biol 109:475-
Muramatsu N, Noson Y. 1971. Purification and characterization of glucose-6-phosphate isomerase from Bacillus stearothermophilus. Arch Biochem Biophy 144: 245-
Nabi IR, Watanabe H, Raz A. 1990 Identification of B16-F1 melanoma autocrine motility-like factor receptor. Cancer Res 50:409-
Nowitzki U, Flechner A, Kellermann J, Hasegawa M, Schnarrenberger C, Martin W. 1998. Eubacterial origin of nuclear genes for chloroplast and cytosolic glucose-6-phosphate isomerase from spinach: sampling eubacterial gene diversity in eukaryotic chromosomes through symbiosis. Gene 214:205-
Riordan JF, McElvany KD, Borders CL. 1977. Arginyl residues: Anion recognition sites in enzymes. Science 195:884-
Rose IA, O’Connel EL. 1961. Intramolecular hydrogen transfer in the phosphoglucose isomerase reaction. J Biol Chem 236:3086-
Schnackerz KD, Noltmann E.A. 1971. Pyridoxal 5’- phosphate as a site-specific protein reagent for a catalytically critical lysine residue in rabbit muscle phosphate isomerase. Biochemistry 10:4837-
Schray KJ, Benkovic SJ, Benkovic PA, Rose IA. 1973. Catalytic reactions of phosphoglucose isomerase with cyclic forms of glucose 6- phosphate and fructose 6- phosphate. J Biol Chem 248:2219-
Schray KJ, HoWell EE. 1979. Anomerization of glucose-6-phosphate : Catalysis by phosphoglucose isomerase. Arch Biochem Biophy 192:241-
Schray KJ, Rose IA. 1971. Anomeric specificity and mechanism of two pentose isomerase. Biochemistry 10:1058-
Seeholzer S. 1993. Phosphoglucose isomerase: A ketol isomerase with aldol C2- epimerase activity. Proc Natl Acad Sci USA 90:1237-
Sun YJ, Chou CC, Chen WS, Wu RT, Meng M, Hsiao CD. 1999 The crystal structure of a multifunctional protein: phosphoglucose isomerase/autocrine motility factor/neuroleukin. Proc Natl Acad Sci U S A 96:5412-
Swenson CA, Barber R. 1971. Proportion of keto and aldelhydo form in solution of sugars and sugar phosphates. Biochemistry 10:3151-.
Tao W, Wang, L, Shen R, Sheng Z. 1989.Complete nucleotide sequences of two phosphoglucoisomerase isozymes from Bacillus stearothermophilus. Nucleic Acids Res 17:10107-
Tor J, Pascual C, Segura RM, Vilaseca J, Schwartz S. 1982 Value of glutathione reductase and glucosephosphate isomerase measurements in infectious diseases. Rev Clin Esp 164:15-
Tor J, Segura RM, Pascual C, Vilaseca J, Guarner ML, Schwartz S. 1981. Determination of serum activity of glucose phosphate isomerase and glutathione reductase in intra and extra hepatic cholestasis. Med Clin (Barc) 77:236-
Walker JI, Layton DM, Bellingham AJ, Morgan MJ, Faik P. 1993. DNA sequence abnormalities in human glucose 6-phosphate isomerase deficiency. Hum Mol Genet 2:327-
Watanabe H, Carmi P, Hogan V, Raz T, Silletti S, Nabi IR, Raz A. 1991. Purification of human tumor cell autocrine motility factor and molecular cloning of its receptor. J Biol Chem 266:13442-
Watanabe H, Takehama K, Date M, Shinozaki T, Raz A. 1996. Tumor cell autocrine motility factors is the neuroleukin/phosphoglucose isomerase polypeptide. Cancer Res 56:2960-
Welch SG. 1971. Qualitative and quantitative variants of human phosphoglucose isomerase. Hum Hered ;21:467-
Willem R, Biesemans M, Hallenga K, Lippens G, Malaisse-Lagae F, Malaisse WJ. 1992 Dual anomeric specificity and dual anomerase activity of phosphoglucoisomerase quantified by two-dimensional phase-sensitive 13C EXSY NMR. J. Biol. Chem 267, 210-
Xu W, Beutler E. 1994. The characterization of gene mutations for human glucose phosphate isomerase deficiency associated with chronic hemolytic anemia. J Clin Invest 2326-
Xu W, Seiter K, Feldman E, Ahmed T, Chiao W. 1996. The differentiation and maturation mediator for human myeloid leukemia cells shares homology with neuleukin or phosphoglucose isomerase. Blood 87:4502-
Yen A, Chiao JW. 1983. Control of cell differentiation during proliferation. I. Monocytic differentiation of HL-60 promyelocytes. Exp Cell Res 146:8
謝嘉榮。1999。人類及大腸桿菌磷酸葡萄糖異構酵素之大量表達系統及純化方法建立。中興大學農業生物科技研究所碩士論文。
林華洋。2000。Bacillus stearothermophilus 6-磷酸葡萄糖異構酵素A中活化中心之酸性胺基酸的功能分析。中興大學農業生物科技研究所碩士論文。
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