定點突變研究Arthrobacter globiformis 組織胺類氧化酵素的特性

Abstract

含銅胺類氧化酵素 [EC 1.4.3.6] 廣泛存於細菌、酵母菌、黴菌、動物及植物界。這一類酵素中，每一個單體包含一個銅離子及一個共價輔因子2,4,5– trihydroxyphenylalanine quinone (簡稱TPQ)。在原核生物中胺類氧化酵素藉由分解胺類來提供微生物生長所需的氮源及碳源; 而在真核生物中所扮演的角色與細胞分化、細胞增生、細胞變性、去毒素作用、傷口的癒合、訊息傳遞及細胞凋亡有關。本實驗所選擇的實驗標的為含銅胺類氧化酵素中的Arthrobacter globiformis histamine oxidase (AGHO)。所有的含銅胺類氧化酵素都具有一段高度保留的蛋白質序列Asn401- Tyr402- Asp403- Tyr404 來扮演其活性中心的角色。 經由目前已知的十七種含銅胺類氧化酵素的蛋白質序列比對，我們對於AGHO上的三個胺基酸位置Y304、K374及T398感興趣，這些位置可能扮演的角色與受質的辨識性、結構的穩定性及酵素的活性有關。其中，Y304的位置被認為是利用短氫鍵與TPQ的O4位置鍵結，並藉此固定TPQ的位置，並且促進TPQ到氧氣分子間的質子轉移。我們將Y304置換成Phe及Glu之後發現，Y304F酵素的活性消失，然而突變後的TPQ生合成並沒有受到影響。藉由本實驗的研究資料顯示，在Y304上的氫氧基缺乏的情況下，活性區內缺乏其他殘基的補償機制，因此，進一步導致二級結構的改變而失去活性。先前認為AGHO的K374的功能為透過單體間的氫鍵來穩定受質的通道，在真核生物中含銅胺類氧化酵素的相對位置上為Arg，而在原核生物的含銅胺類氧化酵素的相對位置上卻是Lys。本實驗將K374的位置置換成為Arg、Cys或His，由酵素動力學的資料顯示，突變株K374R及K374C突變後酵素的Km值及Vmax值產生變化。突變株K374H則與Y304F類似，也由於二級結構的改變而導致活性顯著地下降。T398的位置是位於活性區（catalytic loop）上，並且與D403形成氫鍵鍵結，可能的角色與活性區的穩定及酵素的活性有關。在本實驗中將其取代成為Ser，結果顯示突變株T398S在分解histamine時，仍然具有相當於野生株AGHO 84.3%的催化活性。Y304E突變株具有接近突變前的正常催化活性，並且在於pH 6.5的反應條件下，顯示出高於野生株AGHO的活性。就Circular Dichroism (CD)及分子模擬的結果顯示大部分的突變株其二級結構都有發生變化。由Y304E及T398S的pH profiles及酵素動力學的分析結果發現，其活性區的內在環境可能產生變化。在本實驗中，所有的定點突變並不會去影響到TPQ的生合成，但是突變株與野生株之間，酵素活性仍然存在差異性。

The copper-containing amine oxidases 【EC 1.4.3.6】 are ubiquitiously existed in bacterium, yeast, fungus, plant, and animal kingdoms. They contain a single cupric ion and a covalently bound cofactor, termed 2,4,5– trihydroxyphen- ylalanine quinone (TPQ) in each subunit. In prokaryotes, the amine oxidases allow microorganisms to grow on primary amines. In eukaryotes, they are sugge- sted to play roles in tissue differentiation, cellular proliferation, transformation of cultured cells, detoxification, wound healing, cell signaling, and programmed cell death. Arthrobacter globiformis histamine oxidase (AGHO) is one of a member of copper- containing amine oxidase family with a consensus tetrapeptide seque- nce, Asn401-Tyr402-Asp403-Tyr404, as it’s “active center”. Upon amino acid sequence alignment of 17 known copper containing amine oxidases, we found three amino acid residues in AGHO, Y304, K374, and T398, are interesting due to their potential roles in substrate recognition, structure stab- ility, and enzymatic activity. The Y304 has been proposed to play a central role in anchoring the TPQ via a short hydrogen binding to the O4 of TPQ and in faci- litating proton transfer from TPQ to dioxygen. We changed Y304 into Phe and Glu and find out the catalytic activity of mutants were reduced due to the mutation. The TPQ biogenesis, however, was not altered. Our study reveal the absence of the Y304 hydroxyl group could not be compensated by other residues within the active site. Furthermore, Y304F, an inactive mutant, exhibited an altered secondary structure. The K374 of AGHO (corresponding residue K354 in AGPEO) was suggested to stabilize the substrate channel via an intersubunit hydrogen binding. The residue at this position is usually Arg in the enzymes of eukaryotic origin; while it is Lys in the enzymes of prokaryotic origin. We changed K374 into Arg, Cys, or His. The kinetic data of the K374R and K374C mutants revealed the lower Km as well as lower Vmax values compared with wild type AGHO. Similar to Y304F, K374H mutant was inactive due to the alteration in its secondary structure. The T398 is located at the catalytic loop and form a hydrogen bond with D403. The possible role of T398 may involve in the stability of catalytic loop and, hence, the catalytic activity of the enzyme. We have replaced this residue with Ser. The T398S mutant still has normal catalytic activity by using histamine as substrate (about 84.3% of AGHO activity). Y304E exhibited a near normal catalytic activity, except that it showed a higher enzyma- tic activity at pH 6.5 than that of wild type AGHO. The Circular Dichroism (CD) and the structure modeling show that most mutants showed movement in the secondary structures. The pH profiles and kinetic data of Y304E and T398S showed that the intrinsic environment may be changed due to mutation. In this study, all mutants show no variation in the TPQ biogenesis, but there is distinction in the activity between mutants and wild type AGHO.