溶藻弧菌胺醯組胺酸雙胜肽酶功能性胺基酸於催化特性分析之研究

Abstract

摘要 實驗室從溶藻弧菌 ATCC 17749中選殖出胺醯組胺酸雙胜肽酶(PepD)之基因並解出序列。胺醯組胺酸雙胜肽酶(PepD, EC 3.4.13.3)為胜肽酶家族M20中的一員，可水解雙胜肽L-carnosine及其他特定Xaa-His雙胜肽。此基因之開放讀碼區(ORF)序列共有1473個鹼基對，可轉譯出一條長490個胺基酸的蛋白質，計算其分子量約為53.6 kDa，並且此胺基酸序列和其他弧菌屬之PepD蛋白質序列比對有非常高之相似度。過去對於細菌中胺醯組胺酸雙胜肽酶的研究很少，只針對其序列和部分生化特性進行探討，並無其活性區胺基酸或金屬離子方面相關之研究。根據先前實驗室所架構的pET-28a(+)-pepD質體，表現出N端帶有His-tag之重組蛋白，並利用Ni-NTA親和層析管柱純化。經由序列及結構分析預測溶藻弧菌PepD蛋白上胺基酸位置His80、Asp82、Asp119、Glu149、Glu150、Asp173、His219、Asn260、Arg369、Gly435及His461為活性或受質鍵結區域。將所預測影響金屬鍵結之胺基酸Glu150以及扮演固定His80使其精確的與金屬鍵結的幕後推手Asp82，分別進行定點突變；發現大部分突變蛋白皆失去或降低原有之活性。此外，在預測受質結合位置His219、Asn260、Arg369及Gly435對其做丙氨酸突變試驗，並證明出Arg369為會影響活性之重要胺基酸。而PepD為含金屬之蛋白質，金屬離子對其活性有直接的影響，而其中EDTA可抑制其活性主要原因為失去活性區之金屬；以此為方向置換PepD活性中心金屬為其它二價金屬離子，明確表示出此酵素對金屬的依賴，並且結果為含有不同金屬之PepD具有不同表現的活性。因此，根據本論文實驗結果將提出胺醯組胺酸雙胜肽酶活性區胺基酸之分佈情形與金屬離子扮演重要之功能。

Abstract Earlier investigations in our laboratory, a newly defined aminoacylhistidine dipeptidase from Vibrio alginolyticus ATCC 17749 was characterized via the determination of the corresponding gene sequence. Aminoacylhistidine dipeptidase (PepD, EC 3.4.13.3) is a member of the peptidase family, which catalyzes the cleavage and release of N-terminal amino acid from a dipeptide molecule like L-carnosine (β-Ala-L-His). The pepD gene from Vibrio alginolyticus encodes a polypeptide of 490 amino acids, which has a sequence that is highly similar to that of dipeptidases from various other species. The researches on bacterial PepD were less known and only investigated genetically and biochemically. Previously, the gene was cloned into the pET-28a(+) expression vector and expressed as a (His)6-PepD fusion protein, which was purified via a Ni-NTA column. The sequence alignment and structure model of PepD indicate that His80, Asp119, Glu150, Asp173 and His461 are metal binding site residues, and Asp82, Glu149, His219, Asn260, Arg369, and Gly435 are putative catalysis and substrate binding residues. Site-directed mutations of Asp82, Glu150 and Arg369 residues of PepD exhibit a decline or loss of activity, suggesting that these residues might be involved in substrate and metal binding, thereby dramatically affecting enzymatic activity. The native Zn2+ ion was removed from PepD using an EDTA chelating solution, which resulted in the loss of hydrolysis activity. However, PepD activity could be restored by adding Mg2+, Mn2+, Co2+, Ni2+, Cu2+ or Cd2+, indicating the functional importance of the metal ion. The functional role of these residues in enzyme catalysis and the effect of metal ions will be discussed in this thesis.