標題: Arthrobacter globiformis 組織胺氧化酶受質特異性研究
Research of Substrate Specificity of Arthrobacter globiformis Histamine Oxidase
作者: 楊政剛
Jen-Gong Yang
袁俊傑
Chiun-Jye Yuan
生物科技學系
關鍵字: 氧化酶;受質特異性;組織胺;amine oxidase;substrate specificity;arthrobacter globiformis;histamine
公開日期: 2003
摘要: 含銅胺類氧化酵素 [EC1.4.3.6] 廣泛存在於細菌、酵母菌、黴菌、動物及植物界。此類酵素藉由氧化去胺作用分解一級胺類而產生醛類,氨和過氧化氫。不同來源的胺類氧化酵素彼此之間的受質特異性具有很大的差異。關於受質特異性上的差異一直是研究上的重要課題,為了釐清其中的差異,我們利用AGHO此株酵素來研究,一系列在結構上相似的aromatic amines,在其aliphatic chain 的長度及aromatic ring上一些官能基的修飾,探討其對受質特異性的影響。從其中的kinetic analysis比較中,實驗結果顯示隨著aromatic amines 上帶有胺基的aliphatic chain長度增加,Km會隨之下降。而在一些結構相似的aromatic amine研究之中,發現aromatic ring的hydropathy會影響substrate specificity (Kcat/Km)。 之前結晶結構研究發現,在此類酵素活性區中,存在一個扮演類似閘道的殘基,而在AGHO中,其相對位置為Tyr316。利用定點突變的方式將此位置突變成Ala,His,Glu和Phe。突變株酵素具有正常的TPQ生成能力,但無酵素活性。以Phe取代的話,酵素活性只剩25%。Ala,His或Glu等胺基酸取代,則所產生的酵素不具活性。其中影響的因素還需由進一步的實驗加以探
Copper-containing amine oxidases (CAOs) [E.C.: 1.4.3.6] are ubiquitous in nature, found in bacteria, yeasts, fungi, plants and animals. CAOs catalyze the oxidation of various primary amine substrates to their corresponding aldehydes, with the subsequent release of ammonia and hydrogen peroxide. Substrate preference depends on the enzyme source. AGHO was used as a model to study its substrate preference and kinetics to various amines to elucidate the structural characteristic of amines. The results show that the Km value decreased in nearly linear fashion with increasing chain length of the alkyl carbon chain of amines on aromatic ring. Substrate specificity (Kcat/Km) increased with increasing the hydropathy of the aromatic ring of amines. Y316 of histamine oxidase from Arthrobacter globiformis has been suggested to act as a “gate” to mediate the access of substrate to TPQ. This residue has been replaced with other amino acids such as Phe, Trp, Ala, His, and Glu by site-directed mutagenesis. When Tyr316 was changed to Ala, His, or Glu, the purified recombinant proteins exhibit normal TPQ biogenesis, although their activity are not detectable. When Tyr316 was replaced by Phe, the enzyme was active with a relative activity of around 25% compared of with that wild-type protein using histamine as substrate. These results suggest that Y316 is essential for the enzyme activity of AGHO.
URI: http://140.113.39.130/cdrfb3/record/nctu/#GT009128522
http://hdl.handle.net/11536/56013
Appears in Collections:Thesis


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