Structure and Expression of Mold Crystalline alpha-Glucosidase

霉菌结晶α-葡萄糖苷酶的结构和表达

• 批准号: 04453129
• 负责人: CHIBA Seiya
• 金额: $ 4.1万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04453129/
• 关键词: Complet structure; Disultide bond; Chemical modification; Aspergillus niger; α-Glucosidase

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

1. Asp初级结构的测定。α -葡萄糖苷酶（ANG）：晶体酶（MW, 1.25 × 10^4）是一种含碳水化合物25.5%的糖蛋白，由两个亚基P1 （MW, 3.3 × 10^4）和P2 （NW, 9.8 × 10^4）组成。每个亚基被还原和s -吡啶基化，被几个蛋白酶消化或化学裂解破碎。用Edman法对分离的肽进行测序。根据获得的序列信息确定了两个亚基的一级结构。P1和P2分别由227和719个氨基酸残基组成。在哺乳动物α -葡萄糖苷酶中，P1和P2的c端同源序列是连续的，说明P1和P2是按照P1和P2的顺序合成的单肽链，翻译后与某些蛋白酶裂解形成两个亚基。最近，一个克隆的编码ANG的基因组DNA片段被测序。核苷酸序列表明，两个亚基基因以P1和P2.2的顺序连续存在。CBE对ANG的失活分析及其催化位点的鉴定：以肌醇为原料合成α -葡萄糖苷酶的自杀底物Couduritol B环氧化物（CBE），并对ANG的催化位点进行了检测。CBE对ANG的失活符合准一级动力学。盐酸水解CBE处理过的ANG释放出scyllo-肌醇，表明活性部位的羧酸盐（-OCO）攻击CBE分子的C-2。灭活的ANG用lysc蛋白酶酶切，分离cbe标记肽段并测序。CBE特异性修饰了P2中的Asp-224，这是ANG的催化基团（-COO）。-淀粉酶催化d -材料的水化：我们研究了两种-淀粉酶在^2H_3O中催化d -材料的水化。产物2-脱氧麦芽糖的端粒构型为β型。^2H_2O中反应产物的^2H-NMR分析表明，β -淀粉酶催化d -麦芽糖中双键的顺式水化反应生成β -2-脱氧麦芽糖。ANG糖链结构的测定：P1和P2中有24个修饰氨基酸，无法用Edman法鉴定。其中15个似乎是n-糖基化的，因为每个序列都显示- asn - x - ser /Thr-，除了Ser-296。用n -糖苷酶F对ANG进行处理，高效液相色谱法分离得到7个低聚糖组分。通过核磁共振和成分分析确定了低聚糖的结构。这四种低聚糖中的每一种都含有一个通过α - 1,2键与Man相连的α - d -半乳糖呋喃基残基（Galf）。具有半糖链的三个糖链是新颖的结构。少

项目成果

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N.Shimomura, M.Honma, S.Chiba, S.Tahara and J.Mizutani: "Cysteine-conjugate beta-Lyase from Mucor javanicus" Biosci. Biotech. Biochem.56. 963-976 (1992)

N.Shimomura、M.Honma、S.Chiba、S.Tahara 和 J.Mizutani：“来自爪哇毛霉的半胱氨酸缀合 β-裂解酶”Biosci。

H.Matsui, Y.Tanaka, C.F.Brewer, J.S.Blancard, and E.J.Hehre: "Hydrolysis of alpha-and beta-D-glucosyl fluoride by individual glucosidases ; new evidence for separately controlled "plastic" and "conserved" phases in glycosylase catalysis" Carbohyd. Res.250

H.Matsui、Y.Tanaka、C.F.Brewer、J.S.Blancard 和 E.J.Hehre：“单个葡萄糖苷酶水解 α-和 β-D-葡萄糖基氟化物；糖基化酶催化中单独控制“塑性”和“保守”相的新证据

T.Takayanagi: "Novel Structures of N-Linked High-Mannose Type Oligosaccharides Containing α-Galactofuranosyl Linkage in A.niger α-Glucosidase" Carbohydr.Res.255(in press). (1994)

T.Takayanagi：“黑曲霉 α-葡萄糖苷酶中含有 α-呋喃半乳糖基连接的 N-连接高甘露糖型寡糖的新结构”CarboHydr.Res.255（出版中）。

H.Kawagishi: "Two Lectins from the Marine Sponge Halichondria okadai" J.Biochem.,. 269. 1375-1379 (1994)

H.Kawagishi：“来自海洋海绵 Halichondria okadai 的两种凝集素”J.Biochem.,。