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研究生:鄒延鴻
研究生(外文):Yen-Horng Tsou
論文名稱:探討人類葡萄糖-六-磷酸去氫酵素在其菸鹼醯胺腺嘌呤雙磷酸結合部位鄰近三個精胺酸之生化特性
論文名稱(外文):Biochemical characterization of the three Arginine residues in close proximity to the putative NADP+ binding site of the human glucose-6-phosphate dehydrogenase
指導教授:趙崇義;劉寶瑋
指導教授(外文):DTY Chiu
學位類別:碩士
校院名稱:輔仁大學
系所名稱:生物學研究所
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:1995
畢業學年度:83
語文別:中文
論文頁數:70
中文關鍵詞:葡萄糖-六-磷酸去氫酵素遺傳突變精胺酸酵素活性
外文關鍵詞:G6PDNADP+Arginineenzyme kinetic
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葡萄糖-六-磷酸去氫酵素(Glucose-6-phosphate dehydrogenase; G6PD)
是葡萄糖經由六碳糖磷酸分路(Hexose Monophospate Shunt)代謝的第一
個主要酵素。此途徑的主要生化產物為NADPH和Ribose-5-phosphate,其
中NADPH是保護細胞不易受到氧化損傷(oxidative damage)的重要還原物
質。而G6PD缺乏症大部分是由於G6PD基因上不同的點突變(point
mutation)所引起,且在國人G6PD基因發生點突變的位置中,其G6PD胺基
酸序列454、459、463三個帶正電的精胺酸(Arginine)有較高程度的遺傳
突變產生,這些突變均可能會導致G6PD酵素活性的缺失。近幾年的相關研
究報導指出,在G6PD胺基酸序列386位置的離胺酸,可能為此酵素與基質
NADP+的結合部位,而上述三個帶正電的精胺酸,與此離胺酸的距離十分
接近。故可由此推測,此三個帶正電的精胺酸,可能會影響此酵素與基質
NADP+的結合或其催化能力。在本論文的研究中,利用分子生物學上的技
術,將G6PD胺基酸序列454、 459、463的位置作不同去氧核糖核酸的改變
,而可使得這三個帶正電的精胺酸,改變成其他不同的胺基酸,之後在大
腸桿菌中表達這些不同的 G6PD 蛋白,進而探討這些G6PD突變蛋白對於其
酵素活性的影響。結果發現在G6PD胺基酸序列454位置上的精胺酸對於G6
PD的酵素活性是絕對必須的,任何胺基酸的取代後,均會使得G6PD的酵素
活性幾乎完全的喪失。在G6PD胺基酸序列459位置上的精胺酸,一個帶電
荷的胺基酸對於G6PD的酵素活性是必須的,但經不帶電的胺基酸取代後,
其G6PD的酵素活性便明顯地下降。在G6PD胺基酸序列463位置上的精胺酸
,一個帶正電的胺基酸對於G6PD酵素活性的維持是必須的。由以上實驗結
果可知,上述三個帶正電的精胺酸對於G6PD酵素活性是相當的重要,且可
能扮演了不同的角色。

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme in the
hexose-monophosphate-shunt and its major biochemical function
is to generate NADPH, an important reducing equivalent, for
cells to use. G6PD deficiencies are found mostly due to
diverse point mutation in the G6PD gene associated with loss of
enzyme activity. Previous studies have proposed that Lys386
residue of the human G6PD involves in NADP+ binding. However,
due to the lack of crystalline structure, this putative binding
site of the human G6PD for NADP+ is not well defined. We
hypothesize that the three arginine residues, Arg454, Arg459
and Arg463, which are in close proximity to the putative NADP+
binding region, may play a role in anchoring NADP+ to the
catalytic domain. Mutations in either one of these arginine
positions have been found to be associated with G6PD deficiency
in the Chinese population. By using molecular biological
techniques, human G6PD gene mutated in these arginine residues
has been successfully constructed and overexpressed in E. coli.
Moreover, these mutant human proteins are purified and the
enzyme kinetic studies of these arginine mutants are
undertaken. Our data indicate that Arg454 is a crucial amino
acid for the enzyme function because any substitution in this
position completely diminished G6PD activity. On the other
hand, either a positively or negatively charged amino acid
residue can replaced Arg459 with minimal effect on the enzyme
activity. Only when Arg459 was replaced by neutral amino acid
residue, a significant reduction in enzyme activity was found.
As regard to Arg463, a positively charged amino acid at this
position is essential for G6PD activity. These results
indidcate that the three Arg residues in G6PD are all important
for the enzyme activity. However, their roles in G6PD might be
different.

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