Structure and Expression of Mold Crystalline alpha-Glucosidase

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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