胺醯組氨酸雙胜太活性區域之蛋白質工程研究

Abstract

胺醯組氨酸雙胜肽酶活性區域之蛋白質工程研究 胺醯組胺酸雙胜肽酶(PepD)可催化Xaa-His 雙胜肽而釋放出一中性或疏水性氨基 酸。PepD 酵素通常具有寬廣之受質專一性，包括可水解肌雙胜(carnosine)及相關之加長 肌雙胜(homocarnosine)以及一些三胜肽。加長肌雙胜(homocarnosine)為神經傳導物質γ- 胺基丁酸(GABA)之前驅物及作為GABA 之儲存槽，因而可作為抗癲癇之治療用途。雙 胜肽酶因其可將加長肌雙胜水解成GABA 以及其可對加長肌雙胜之儲存調控，將可作 為神經保護藥物開發之標的而值得研究。另外，雙胜肽酶亦具有可作為抗細菌治療、以 及癌症治療之標的等應用之潛力。 本實驗室先前已由溶藻弧菌中選殖出一可水解雙胜肽之PepD。所表現純化之PepD 可分解肌雙胜(carnosine)及相關之加長肌雙胜(homocarnosine)，但不分解三胜肽。經由對 胺醯組胺酸雙胜肽酶之生化特性測定、活性區域氨基酸之確認、以及X-光晶體結構測 定，我們已獲得初步之酵素結構-功能-反應機制之關係。然而酵素之水解機制仍未被完 全了解。在本計畫中、我們將進一步針對酵素-抑制劑複合物進行晶體結構鑑定，以進 一步了解酵素之結構-反應活性之關係。同時，我們也將針對可能與受質鍵結之氨基酸 進行定點飽和突變之蛋白質工程、篩選調控對肌雙胜或加長肌雙胜受質具專一性之變 種、測定其對受質結合動力學之影響，以增進或擴展酵素之活性或專一性。同時，我們 也將利用EXAFS 和NMR 技術研究氨基酸突變對金屬鍵結環境之影響。最後，我們也 將結合胺醯組胺酸雙胜肽酶酵素與單株抗體建構抗體導向酵素藥物，針對神經傳導物質 進行導向式催化，以測試其作為神經保護或抗顛癇藥物開發之標的。因此，經由本計畫 之執行，我們將對酵素結構-功能-反應機制之關係有更進一步的了解，同時也可加速開 發抗體導向之神經保護及抗癲癇之酵素前導藥物。

The Research on Protein Engineering of Aminoacylhistidine Dipeptidase Active Site Aminoacylhistidine dipeptidase (PepD) catalyzes the cleavage and release of an N-terminal amino acid, which is usually a neutral or hydrophobic residue, from a Xaa-His dipeptide or degraded peptide fragments. The enzyme also exhibits broad substrate specificities towards unusual dipeptides carnosine (β-Ala-L-His) and homocarnosine (γ-aminobutyl-His) as well as a few distinct tripeptides. Homocarnosine is the precursor and reservoir of γ-aminobutyric acid (GABA) and being used in anti-seizure therapy. Therefore, the enzyme capable of hydrolyzing homocarnosine into GABA and regulating homocarnosine holds great potential in acting as target of neuroprotective agent development. In parallel, the PepD enzyme has also been shown as target for anti-bacterial treatment and as therapeutic agent for cancer therapy. We have previously cloned and overexpressed a PepD enzyme from Vibrio alginolyticus. The purified PepD enzyme exhibited substrate specificity to carnosine and related homocarnosine, but not to tripeptide. Biochemical and molecular biological as well as structure determination of PepD have also identified several amino acid residues involved in metal- or substrate-binding. However, the preliminary aspects of the enzymatic hydrolysis mechanism are still ambiguous. In this proposal, we will continue to determine the X-ray structure of PepD-inhibitor complex, to enhance or expand enzyme activity and specificity through active site combinatorial mutagenesis, to select PepD mutant with higher activity towards homocarnosine, and to determine the enzyme-substrate dynamics. In parallel, the mutational effect on putative metal binding environments will be studied using EXAFS and NMR techniques and compared with that of wild-type PepD. Finally, cancer specific antibody will be conjugated with PepD to construct the potential antibody-directed enzyme therapy agent for the development of mAb-directed neuroprotective or anti-seizure prodrug. Thus, the execution of this project will advance our understanding of the structure-activity-mechanism relationships of aminoacylhistidine dipeptidase in general and facilitate the development of antibody-directed neuroprotective or anti-seizure enzyme therapy.