利用定點突變方法以及突變株與受質複合體結構驗證人類麩醯胺環化酵素之催化機轉

麩醯胺環化酵素在負責將胜肽鏈N端的麩醯胺轉環為環化麩胺酸的工作中扮演一個很重要的角色。這個步驟在許多的賀爾蒙或是蛋白質中都是很重要的。舉例來說，例如甲促素刺激素或是神經張素，這些酵素都需要麩醯胺環化酵素在它們蛋白質的生成過程中的最後一個步驟將它們N端的麩醯胺轉化為環化麩胺酸，如此這些酵素才具有正常的功能與活性。在之前的研究發現，麩醯胺環化酵素已經在許多的物種之中被發現，譬如豬、牛或是木瓜乳液等，同時也包括人類。但是，有關於麩醯胺環化酵素的結構與催化機轉仍然是一個未知的謎題。來自人類的麩醯胺環化酵素已經成功的被轉殖、表現與純化到大量並且具有高純度的人類。同時間，此酵素進行基本的活性測定，證明此酵素是具有麩醯胺環化酵素的活性。之後黃學長又成功的利用X光繞射原理將人類麩醯胺環化酵素的結構與酵素/抑制物的共結晶結構解出來。從以上的結果配合結構資料庫的結構比對的結果，初步的催化反應機制可以被預測出來。為了要更清楚的了解人類麩醯胺環化酵素的催化反應機轉，有一些關鍵的殘基可以被推選出來，例如第201號位置的麩胺酸、第248號與第305號的天門冬氨酸、第304位置的麩醯胺、第307與319號的組胺酸、第325號的苯丙胺酸以及第329位置的色胺酸。這些殘基都距離中心活性區域相當的接近，有可能對酵素的催化或是基質與酵素的結合有相當大的關係。由此可見，我們可以利用定點突變的方法將這些殘基突變為旁鏈結構相似或是不相似的殘基，並且利用偶聯酵素活性測定法將這些突變株的活性測定出來。此外，高效液相層析法被用來證明測量幾個不同胺基酸長度的賀爾蒙對於非突變型的人類麩醯胺環化酵素的反應效率。從偶聯酵素活性測定中了解，第201號位置的麩胺酸如果突變為麩醯胺反應活性幾乎消失，因此可以利用此突變株的晶體與基質結合反應，並解出此突變株與基質的複合體結構。這些突變株的活性測定以及突變株與基質的複合體結構可以清楚的驗證先前黃學長所推測之人類麩醯胺環化酵素的催化反應機轉，可以讓此一反應機轉變得更有利而且讓人信服。

關鍵字

麩醯胺環化酵素；催化機轉； X-光結晶學；定點突變

並列摘要

Glutaminyl cyclase (QC) is a critical enzyme that is responsible for the conversion of N-terminal glutamine into pyroglutamic acid, which is an important event during protein synthesis. Many hormones, such as thyrotropin-releasing hormone and neurotensin, need QCs for modifying their N-terminal glutamine and subsequently become an active form. In the previous studies, QCs from various species, such as porcine, bovine, papaya or human have been cloned, expressed and characterized. However, the exact catalytic process still remained ambiguous due to the lack of ingormation on QC structure. The human QC has been cloned and expressed in large scale and purified successfully by Kai-Fa Huang in our group. He also determined the first QC and QC/inhibitors structures by x-ray crystallography (2005 in press). Enzyme catalytic mechanism was proposed preliminarily based on the structural alignment and the solved structures. In order to confirm the catalytic mechanism proposed by Huang, some key residues, like E201, D248, D305, Q304, H307, H319, F325 and W329, which are likely to involve in the catalytic mechanisms or play vital roles in the substrate recognition, have been chosen for site-directed mutagenesis. The catalytic ability of wild-typed human QC was assessed and characterized by high performance liquid chromatography. Coupled enzyme assay was used to evaluate the activities of the mutants. Among them, E201Q, which dramatically lost its catalytic activity, was subjected to further investigation. The E201Q/glutamine-t-butyl ester complex structure was determined. The structural information of substrate binding is supportive to the previous suggestion on enzyme catalysis. Combined with results from the biological activity analysis of human QC mutants, the proposed mechanism could be more solid and reliable.

並列關鍵字

glutaminyl cyclase ； catalytic mechanism ； X-ray crystallography ； site-directed mutagenesis

參考文獻

Hinke, S.A., Pospisilik, J.A., Demuth, H.-U., Mannhart, S., Kuhn-Wache, K., Hoffmann, T., Nishimura, E., Pederson, R.A. and McIntosh, C.H.S. (2000) Dipeptidyl peptidase IV (DPIV/CD26) degradation of glucagon. J Biol Chem, 275, 3827-3834.

Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K. and Walter, P. (2002) In Molecular Biology of the Cell. 4th ed. Garland Publishing, New York, USA, pp. 299-374.

Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K. and Walter, P. (2002) In Molecular Biology of the Cell. 4th ed. Garland Publishing, New York, USA, pp. 689-702;713.

Bateman, R.C., Jr., Temple, J.S., Misquitta, S.A. and Booth, R.E. (2001) Evidence for essential histidines in human pituitary glutaminyl cyclase. Biochemistry, 40, 11246-11250.

Bzymek, K.P. and Holz, R.C. (2004) The catalytic role of glutamate 151 in the leucine aminopeptidase from Aeromonas proteolytica. J Biol Chem, 279, 31018-31025.

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利用定點突變方法以及突變株與受質複合體結構驗證人類麩醯胺環化酵素之催化機轉

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