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研究生:許勝鈞
研究生(外文):Hsu, shung cheng
論文名稱:Staphlococcusepidermidis和Staphylococcushyicus脂肪脢之結構,功能與電腦模擬
論文名稱(外文):The structure,function and computer modeling of Staphlococcus epidermidis and Staphylococcus hyicus lipases
指導教授:蕭介夫蕭介夫引用關係
指導教授(外文):Jei-fu Shaw
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:生物技術研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:1996
畢業學年度:84
語文別:中文
論文頁數:68
中文關鍵詞:脂肪脢單點突變
外文關鍵詞:lipasesite-direct mutagenesis
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中文摘要 已
知三種 Staphylococcus 脂肪的基酸序列,其彼此 相似
性約70%,因此利用單點突變的方式來探討結構和功能的 關係,
以便改變基質特異性或增加催化速率。 從己知
結構的脂肪得知其三級結構有其相似之處,在活 性中心
Serine 之前為b-strand之後為a-helix而Serine必位於 5個共同
基酸(Gly-X-Ser-X-Gly)所形成彎曲之中.其活性中 心Serine之
後基酸的基上的氫能提供氫鍵穩定陰氧離子, 而當酵素和受
質脂類結合,其受質醇基部分會和b-strand 平行 接觸,而醯基部
分朝向a-helix,而a-helix能提供能量來穩定在 催化反應過程中所
形成的過渡四面體. 在Staphlococcus
epidermis 脂肪中在b -eSer( 418 ) -a 結構中的針對b-
strand 中Leucine414 構築L414F, L414V, L414I三個突變基
因,於a-helix 中isoleucine424構建4個突變 的基因I424V, I424
A, I424S, I424G於彎曲結構中將Histidine 417突變成glutamine.
結果發現L414V, L414I 完全失去活性, L414F由電泳片活性染色
顯示只有些微活性,可見Leucine 414 在受質結合有一定的影響,
然而I424V 對受質催化速率都比 wild type 高,但對p-
nitrophenyl caprate 有較低結合能力卻有 較高的催化常數. I424S
對不同受質的催化速率都比wild type 低,而其對p-
nitrophenyl butyrate 有較高結合能力,在以 p-nitro phenyl
caprate 為受質時,發現其kcat比wild type 高, 可見由於L424S 會
造成構形的變化。至於其他酵素I424A, I424G, H417Q 完全
失去活性,乃由於突變造成結構破壞. 在Staphlococcus
hyicus 脂肪中將活性中心Ser(369)之 後的Met370 突變成
Ala, Leu,但皆沒有活性,可見突變後造成 無法有效提供氫原子
給受質和酵素,無法穩定催化四面體形 成。

直接以Pseudomonas glumae脂肪的蛋白質骨架作為
Staphlococcus epidermis 脂肪骨架,再以區域突變的方式將 活
性中心附近的基酸置換成Staphlococcus epidermis脂 肪的
基酸,然後比較wild type 和 I424S 結合及催化作用中 CHARM
energy 的差異,可知I424能參與穩定催化反應中催 化四面體
形成.

Abstract

Three Staphylococcus lipases genes were cloned by PCR technique
and successfully overexpressed in E.coli(DE3) using the T7 RNA
polymerase expression system. The similarities of homology
between these three Staphylococcus lipases were
approximately 70%. In order to elucidate the
relationships of structure and function in these lipases, we
use site-directed mutagenesis technique to modify substrate
specificity of the enzymes and hope to increase catalytic
rate. All lipases show
significant similarities in their three-dimensional
structures from the known crystal structure in lipases. In all
of them the catalytic serine is embedded in a signature
pentapeptide sequence Gly-X- Ser-X-Gly and is located in
a unique supersecondary structure at a tight bend
between a b-strand and a-helix. The oxyanion hole of each is
proposed to involve the backbone NH group of the residue
immediately C-terminal to the active site serine.
When an ester bind in their active site so that the
alcohol portion rest on a floor formed by the end of b-
strand which was predicted to be involved in substrate binding ,
while the acyl chain points toward the a-helix and
provides the a helix dipole moment to stabilize the
tetrahedral intermediate during the catalytic
reaction.

In Staphlococcus epidermis lipase gene, we constructed three
mutants(L414F, L414I, L414V) located in the b- strand near
active site Serine418, one mutant(H417Q)at a tight
bend next to Ser 418, four mutants(I424V, I424G,
I424A, I424S) in the a- helix. Mutants Leu414 and
L414V completely lost enzyme activity and Leu414F showed a
little activity from gel activity staining. I424V
increase kcat/Km value when p-nitrophenyl butyrate or
p-nitrophenyl caprate was use as a substrate, it showed
decrease substrate binding affinity for p-nitrophenyl caprate
but show higher kcat toward p-nitrophenyl caprate. The
I424S mutant enzyme had decrease kcat/Km value
when p-nitrophenyl butyrate or p- nitrophenyl
caprate was employed as a substrate, but it showed higher
binding affinity than wild type toward p-nitrophenyl butyrate
and higher kcat value than wild type when using p-
nitrophenyl caprate as a substrate . Possibly due to the
denaturation of their local structural motif, the mutant
proteins(I424A, I424G, H417Q)completely lost enzyme activity

In Staphlococcus hyicus gene, we constructed two mutants (M370L,
M370A). Substitution of Met370 , which was next to active site
Serine369, with leucine or alanine lost enzyme activity possibly
due to the disruption of oxyanoion hole in stabilizing the
transition state of the rate- limiting step of the reaction
mechanism of these enzymes. In order to
model Staphlococcus epidermis lipase protein backbone
using Pseudomonas glumae lipase as a template, was replaced the
active site the conserved region of active by "mutant
range ". Using p- nitrophenyl butyrate as a
substrate, the differences of binding and catalysis CHARM
energy support that I424 is more important for catalysis than
substrate binding and it is involved in stabilizing tetrahedral
intermediate.



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