Structure and Expression of Mold Crystalline alpha-Glucosidase

1.天冬氨酸一级结构的确定。尼日尔α-葡萄糖苷酶(Ang)：结晶酶(Mw，1.25×10^4)是一种糖蛋白，含25.5%的碳水化合物，由两个亚基组成：P1(Mw，3.3×10^4)和P2(NW，9.8×10^4)。每个亚基被还原和S-吡啶甲基化，被几次蛋白酶消化或化学切割而碎裂。用Edman‘s方法对分离的多肽进行测序。根据获得的序列信息确定两个亚基的一级结构。P1和P2分别由227个和719个氨基酸残基组成。哺乳动物α-葡萄糖苷酶P_1和P_2 C-末端的同源序列是连续的，表明P_1和P_2是以P_1和P_2的顺序合成的单链多肽链，翻译后与某些蛋白酶切割形成两个亚基。最近，一个编码血管紧张素转换酶基因的克隆DNA片段被测序。该核苷酸序列指示在…进一步分析表明，两个亚基基因按P1和P2.2的顺序连续定位。以肌醇为原料合成了α-葡萄糖苷酶的自杀性底物--环氧化香豆醇B(CBE)，并对其催化部位进行了测定，结果表明CBE对Ang的失活符合一级反应动力学。CBE处理的Ang的HCl水解物释放了肌醇，提示活性中心的羧酸(-OCO)攻击了CBE分子的C-2。用Lys-C酶消化灭活的血管紧张素转换酶，分离和测序CBE标记的多肽。CBE对ANG的催化基团P2中的Asp-224进行了特异性修饰。β-淀粉酶催化D-金属水合反应：我们考察了两种β-淀粉酶在^2H2O中催化的D-金属水合反应。^2H_2O中反应产物的~2H-核磁共振分析表明，β-淀粉酶催化D-麦芽醛的双键顺式水合生成β-2-脱氧麦芽糖。血管紧张素转换酶糖链结构的测定：P_1和P_2中有24个修饰氨基酸，用Edman‘s法无法鉴定。其中15个似乎是N-糖基化的，因为除Ser-296外，每个序列都显示-Asn-X-Ser/Thr-。用N-糖苷酶F处理Ang，用高效毛细管电泳法分离得到7个低聚糖组分，其结构经1-核磁共振和成分分析确定。这四种低聚糖中的每一种都含有α-D-呋喃半乳糖基残基(GALF)，通过α-1，2-键与人相连。含有GALF的三条糖链是一种新颖的结构。较少

1. Determinatin of primary structure of Asp. niger alpha-glucosidase (ANG) : The crystalline enzyme (MW, 1.25 x 10^4) is a glycoprotein containing carbohydrate in 25.5%, which is composed of two subunits, P1 (MW, 3.3 x 10^4) and P2 (NW, 9.8 x 10^4). Each subunit, which was reduced and S-pyridylcthylated, was fragmented by several protease digestions or chemical cleavage. The isolated peptides were sequenced by Edman's method. The primary structurs ob both subunits were determined from the sequence information obtained. P1 and P2 were composed of 227 and 719 amino acid residues, sequence respectively. The homologous sequence in the regions of C-terminal of P1 and P2 are continuous in mammal alpha-glucosidases, suggesting that P1 and P2 are synthesized as a single peptide chain in the order of P1 and P2, and that after translation two subunits are formed by cleavage with some proteases. Recently a cloned genemic DNA fragment coding ANG have been sequenced. The nucleotide sequence indicat … More ed that two subunits genes were continuously located in the order of P1 and P2.2. Analysis of inactivation of ANG with CBE and identification of its catalytic site : We synthesized Couduritol B epoxide (CBE), a suicide substrate of alpha-glucosidase, from myo-inositol and examined the catalytic site of ANG.The inactivation of ANG with CBE followed psedo-first-order kinetic. HCl hydrolysis of CBE-treated ANG gave the release of scyllo-inositol, suggaesting that carboxylate(-OCO) in active site attackes the C-2 of CBE molecule. The inactivated ANG was digested with Lys-C protease, and CBE-labeled peptide was isolated and sequenced. CBE specifically modified Asp-224 in P2, which was the catalytic group (-COO)of ANG.beta-Amylase-catalyzed hydration of D-matal : We examined D-matel hydration catalyzed by two beta-amylase in ^2H_3O.The anomeric configuratin of the product, 2-deoxymaltose, was determined to be beta-type. The ^2H-NMR analysis of reaction product in ^2H_2O indicated that beta-amylase catalyzed the cis hydration of the double bond in D-maltal to form beta-2-deoxymaltose.4. Determinatin of sugar chain structure of ANG : There are 24 modified amino acids in P1 and P2, which cannot beidentified by Edman's method. Fifteen of them seemed to be N-glycosylated, because each sequence showed -Asn-X-Ser/Thr-, except for Ser-296. ANG was treated with N-glycosidase F, and seven oligosaccharide fractions were isolated by HPLC.The structures of the oligosaccharides were determined by ^1-NMR and compositional analysis. Each of the four oligosaccharides contains and alpha-D-galactofuranosyl residue (Galf) linked to Man via an alph-1, 2-linkage. Three suger chains having Galf are novel structures. Less