甜杏仁葡萄糖甘酵素之受質專一性與其抑制作用的研究

Abstract

甜杏仁中含有數種葡萄糖甘同功酵素，常見的為雙股醣蛋白 AA、AB、BB 。本實驗室已幸運的從粗純品中，經 Protein -Pak 管柱分離出一種單分 子 (monomeric) 同功酵素，其分子量為 39,000，等電點 (pI) 為 4.55。為充分了解此同功酵素之特性，本論文研究其受質之專一性、酸鹼 度對其活性之影響及抑制作用。由六種結構類似之受質與酵素在不同酸鹼 性下反應之 pH -profile 顯示，酵素之催化活性至少由兩個胺基酸所調 控，而真正具有催化活性之物種為單質子化 (monoprotonated, EH) 之酵 素分子。其 pK1 值因受質不同而異，約介於 3.5 - 4.7 之間，而 pK2 則介於 6.1 -6.5 之間，第一個 pKa 變化較大，顯示酵素活性位置中， 非醣基結合副位較不明確；而第二個 pKa 可能為同一胺基酸。另外， PNPG 與 PNPGal 作用所顯示的 pK1 與 pK2 值極為相似，再加上抑制作 用之研究，顯示 PNPG 與 PNPGal 結合在同一活性位置上，此結果與 Walker 和 Axelrod 所提之單一活性位置論相符。由各種受質之反應性研 究顯示，受質醣基 C5 上有 CH2OH基時可促使催化反應進行，但會阻礙受 質與酵素的結合。 C4 上之 OH 基於 a 位置時有利於受質與酵素之結合 ，且若 C5 上有 CH2OH 基時，C4 上之 OH 基於 a 位置時有利於催化反 應；若 C5 上無 CH2OH 基時，C4 上之 OH 基於 a 位置時反而不利催化 反應。而苯環上之硝基取代基的位置，對反應性與結合性均無明顯的影響 。由抑制作用之研究顯示 w-N-苯甲醯組織胺為其最佳抑制劑，而任何增 長咪唑基與苯基間之鍵長者均使其結合性下降。而任何在苯基上附加之取 代基均使抑制效果下降。顯示酵素活性位置中之醣基副位與非醣基副位間 之最適距離為相當於 -C(O)NHCH2CH2- 之鍵長，而非醣基副位之空間大小 恰可容一苯基. An isozyme of b-glucosidase was purified from commercial crude enzyme (Sigma Co.25000 units, G-0395, Lot70H4069). The purified protein has been characterized to be monomeric glycoprotein with molecular weight of 39,000 and pI 4.55 that is distinguished from reported isozymes such as A, B, AB. In order to understand the catalytic function of this enzyme, the studies of substrate specificity, pH-dependence and inhibition were performed. Six substrates including o-nitrophenyl-b-D- gluco pyranoside (ONPG), p-nitrophenyl-b-D-glucopyran oside (PNPG), o-nitrophenyl-b-D-galactopyranoside (ONPGal), p- nitrophenyl-b-D-galactopyranoside (PNPGal), p-nitrophenyl-b-D- xylopyranoside (PNPX), p-nitrophenyl-a-L-arabinopyranoside (PNPA) were hydrolyzed by the enzyme in various of pH. All pH- profiles show a bell-shaped curve feature. Suggest that two apparent pKas involve in the catalytic hydrolysis. The first pKa values are in the range of 3.5-4.7 while the second pKas appear at the values around 6.3 ± 0.2. In comparison with PNPG and PNPGal, the high similarity of their pH-profiles indicates that PNPG and PNPGal bind to the same active site of the enzyme. This is consistent with the argument of Walker and Axelrod [D. E. Walker, and B. Axelrod (1978) Arch. Biochem. Biophys., 187, 102-107.]. From the study of substrate specificity, we concluded that (1) the CH2OH group on the C5 of glycone moiety of substrate promotes the catalytic reaction while it attenuates the binding affinity of substrate with the enzyme, (2) the a-configuration of OH group on the C4 of glycone portion of substrate strengthens the binding of substrate to the enzyme. The inhibition was also studied extensively. Results show that w-N-benzoylhistamine is the best inhibitor tested with Ki = 0.04 uM. Any increment of the chain length between phenyl group and imidazole reduces the binding power of inhibitor with enzyme. Adding subst ituent to the phenyl group of inhibitor results in increasing Ki value.