醯亞胺水解酶之金屬數目：脊椎動物與微生物間之差異

Abstract

醯亞胺水解酶為一可催化眾多環狀醯亞胺水解反應之酵素。在生理上，它參與了嘧啶代謝過程中的第二步驟，將環狀的二氫嘧啶水解開環。而對於醯亞胺水解酶來說，位於其活性中心的金屬輔因子扮演了相當重要的角色。在本篇論文中，我們首先將探討不同來源醯亞胺水解酶的金屬選擇性及其影響，不論來源是從脊椎動物或是微生物，過渡金屬的中的二價鈷離子與二價鋅離子具有較高的活性，但其他的二價離子同樣能與催化中心相結合。結構的研究顯示在三種不同狀態下脊椎動物的醯亞胺水解酶，是其催化中心內不含有金屬輔因子、含有一個金屬輔因子以及含有兩個金屬輔因子。它的整體結構均保有一(□□□)8-TIM型主結構以及一個小的□片層型結構，催化中心內的六個胺基酸也都有被保留。然而比較這些結構發現，隨著催化中心與金屬輔因子結合後，位於外層的□8-□1區域也隨之變動而縮減了受質跟產物出入的通道大小。此種由於金屬輔因子鍵結所誘導而造成區域的構型變化是第一次被報導發生在醯胺水解酶(amidohydrolase)上，且很可能是醯亞胺水解酶，尤其是脊椎動物來源的所特有之現象。最後，本篇論文利用蛋白質體技術，發現了台灣數種刺桐在遭受刺桐釉小蜂造癭現象後，出現了影響光合作用和微生物入侵刺桐的現象，可能為刺桐成癭後所產生的大量死亡的原因。

Imidase (EC 3. 5. 2. 2) catalyzes the hydrolysis of a variety of cylic-imides. It also catalyzes the second step of the reductive pyrimidine degradation, the reversible hydrolytic ring opening of dihydropyrimidines. Metal binding in active site is the most important cofactor in imidase. In this thesis, the metal selectivity of imidasewas confirmed. Imidase that obtained from either bacterial or vertebrate, contains transition metal ions, Co2+, Zn2+ showed the higher enzyme activity, but other divalent ions are also inserted. Furthermore, the structures of apo-imidase, mono-metal imidase and di-metal imidase of T. nigroviridis origin all consisted of a core (□□□)8 TIM-barrel fold and a small □-sheet domain, and the catalytic active sites consist of six highly conserved amino acid residues. Upon the metal binding, the outward loop □8-□1 moved toward active site and decreased the width of tunnel passage for substrates and products. This metal induced conformational changes in dynamic loop is first described in imidase and might only existed in vertebrate. The structural informationa explains the long standing question as why the metal requirement varied among imidase from vertebrate and microorganism. Finally, the tools of proteomics were first used to study the protein changes at the early stage of the erythrina gall development. The results indicated that the plant photosynthetic reaction was interrupted and proteins from invaded exotic microorganism were abundant in Erythrina leaves. The interactions of insect, plant, microorganism and environmental changes may work together to induce this ecological disaster.