Title

探討人類粒線體蘋果酸酶之Arg67反丁烯二酸位點突變酶對結構之影響

Translated Titles

Structural Studies of Arg67 Fumarate-Site Mutants of Human mitochondrial NAD(P)+-Dependent Malic Enzyme

Authors

陳韋霖

Key Words

蘋果酸酶 ; 結構 ; malic enzyme ; structure

PublicationName

中興大學生命科學系所學位論文

Volume or Term/Year and Month of Publication

2018年

Academic Degree Category

碩士

Advisor

洪慧芝

Content Language

繁體中文

Chinese Abstract

人類粒線體蘋果酸酶 (human mitochondrial NAD(P)+–dependent malic enzyme, m-NAD-ME, ME2),是一種同源四聚體 (homotetramers) 結構蛋白。人類粒線體蘋果酸酶為蘋果酸酶 (malic enzyme) 的三種異構型 (isoform) 中的一種,ME2相比細胞質蘋果酸酶 (cytosolic NAD(P)+–dependent malic enzyme, c-NAD-ME, ME1),人類粒線體蘋果酸酶具有異位調節區 (allosteric site) 與exo-site,ME2可藉由異位調節區與反丁烯二酸 (fumarate) 結合來調控其酵素活性。人類ME2結構構型還有四種不同型態,開放型 I、II (open form I、II),關閉型 I、II (closed form I、II),會依照fumarate與substrate結合而有型態的變化。為了探討人類ME2重要位點突變酵素與野生種 (WT) 的酵素活性差異,將先前研究ME2重要異位調節區突變酵素 (allosteric site mutants) R67A、L79F、I88L和Y175N及重要單核苷酸多態性突變酵素 (Single Nucleotide Polymorphism, SNP) 的R67Q和Q89P進行結晶實驗,藉由結構方面來進一步探討這些重要突變株與SNP為何有酵素活性上相對於野生種有明顯差異。在酵素活性極低的R67A、R67Q和I88L中,R67A和R67Q其構型有從closed form轉變open form的現象,而I88L構型則無太大變化;在酵素活性較低,但會被fumarate活化的Y175N,其構型並無太大變化;而在酵素活性較低,但會被fumarate活化的L79F和Q89P,雖然有結晶形成,但卻沒有較高的電子訊號,因此無結構數據。而藉由R67A、R67Q、I88L、Y175N相互進一步探討,ME2構型變化與酵素活性的影響。

English Abstract

Human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME, ME2) is a homotetramers structural protein. ME2 is one of three isoforms of malic enzyme. Human mitochondrial NAD(P)+-dependent malic enzyme compared to Human cytosolic NAD(P)+-dependent malic enzyme (c-NAD-ME, ME1) has allosteric site and exo-site. ME2 can regulate its enzyme activity by binding an allosteric regulatory of fumarate. There are four different types of structural configurations. Open I, II, and closed I, II will change according to the combination of fumarate and substrates. In order to investigate the difference in enzyme activity between human ME2 allosteric site mutants and wild type (WT), R67A, L79F, I88L and Y175N and the important single nucleotide polymorphism mutation R67Q, Q89P, was studied. By crystallization experiments, and use there crystal structure to further explore why these important enzymes have significant differences in enzyme activity when compared to WT. R67A, R67Q and I88L have very low enzyme activities. The structure of R67A and R67Q transit the closed form to the open form. I88L structure had no significant. The enzyme activity of Y175N is low, but it is activated by fumarate, and its configuration does not change significant; The structure of L79F and Q89P do not have higher electron signals for resulting the structure-related data even the crystal formation. The effects of ME2 structure changes and enzyme activity were explored by mutants of R67A, R67Q, I88L and Y175N.

Topic Category 生命科學院 > 生命科學系所
生物農學 > 生物科學
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