溶藻弧菌胺醯組胺酸雙胜肽酶之生化特性分析及其功能性胺基酸之研究

Abstract

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。胜肽酶家族M20中的酵素皆屬於金屬雙胜肽酶，而經由研究發現可被應用於抗菌、癌症的臨床治療及神經傳導物質的調控等方面。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，並無其生理角色或活性區胺基酸相關之研究。本論文將溶藻弧菌pepD基因殖入pET-28a(+)質體中，表現出N端帶有His-tag之重組蛋白，並利用Ni-NTA親和層析管柱純化之。純化出的蛋白質對於特定的Xaa-His雙胜肽(例如: L-carnosine)具有水解的能力，但無水解三胜肽之活性。經酵素動力學研究，溶藻弧菌PepD蛋白對雙胜肽L-carnosine之Km與kcat值分別為0.36 mM與8.6 min-1。經由序列分析預測溶藻弧菌PepD蛋白上胺基酸位置His80、Asp82、Asp119、Glu149、Glu150、Asp173及His461為活性區胺基酸。其中將所預測影響金屬鍵結之胺基酸Asp119以及扮演催化角色之胺基酸Glu149分別進行飽和定點突變，發現大部分之突變蛋白皆失去或降低原有之活性。此外，以同屬M20胜肽酶家族之PepV蛋白結晶結構為模板做出溶藻弧菌PepD蛋白之同源模擬，顯示出相同之活性區胺基酸。因此，根據本論文實驗結果將首次提出胺醯組胺酸雙胜肽酶活性區胺基酸之分佈情形與其可能扮演之功能。

Proteins of the aminoacylase-1/metallopeptidase 20 (Acyl/M20) family were characterized to contain a zinc-binding domain at their active site. Aminoacylhistidine dipeptidase (PepD, EC 3.4.13.3) is a member of peptidase family M20 which catalyzes the cleavage and release of N-terminal amino acid, usually are neutral or hydrophobic residue, from Xaa-His peptide or polypeptide. We have cloned a PepD gene, which shared high sequence identity with PepD from various Vibrio spp. and 63% from Escherichia coli and Salmonella typhimurium, from Vibrio alginolyticus. V. alginolyticus PepD was expressed and purified by Ni-NTA column. The kinetics values including kcat (8.6 min-1), kcat/Km (0.398 mM-1s-1) of bacterial PepD were first identified. Sequence analysis revealed that Asp82 and Glu149 were probable active site residues and that Asp119, Glu150, Asp173, and His461 were probable metal ion binding residues of V. alginolyticus PepD. Site-directed mutations of D119 (putative metal ion binding site residue) and E149 (putative active site residue) residues of PepD exhibited activity decreasing or losing, as compared with wild-type PepD. The homology model of V. alginolyticus PepD, based on that of L. delbrueckii PepV structure, exhibited similar active site pocket as predicted. The functional role of these residues on enzyme catalysis and kinetics will be discussed in the thesis.