Active site mapping of amylo-alpha-1,6-glucosidase in porcine liver glycogen debranching enzyme using fluorogenic 6-O-alpha-glucosyl-maltooligosaccharides

Glycogen debranching enzyme (GDE) has two enzymatic activities, 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase. Products with 6-O-alpha-glucosyl structures formed from phosphorylase limit dextrin by the 4-alpha-glucanotransferase activity are hydrolyzed to glucose by the amylo-alpha-1,6-glucosidase activity. Here, we probed the active site of amylo-alpha-1,6-glucosidase in porcine liver GDE using various 6-O-alpha-glucosyl-pyridylamino (PA)-maltooligosaccharides, with structures (Glcalpha1-4)(m)(Glcalpha1-6)Glcalpha1-4(Glcalpha1-4)(n)GlcPA (GlcPA, 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol residue). Fluorogenic dextrins were prepared from 6-O-alpha-glucosyl-alpha-, beta-, or gamma-cyclodextrin through partial acid hydrolysis, followed by fluorescent tagging of the reducing-end residues of the hydrolysates and separation by gel filtration and reversed-phase HPLC. Porcine liver GDE hydrolyzed dextrins with the structure Glcalpha1-4(Glcalpha1-6)Glcalpha1-4Glc to glucose and the corresponding PA-maltooligosaccharides, whereas other dextrins were not hydrolyzed. Thus, substrates must have two glucosyl residues sandwiching the isomaltosyl moiety to be hydrolyzed. The rate of hydrolysis increased as m increased and reached maximum at m = 4. The rates were the highest when n = 1 but did not vary much with changes in n. Of the dextrins examined, Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-6)Glcalpha1-4Glcalpha1-4GlcPA (6(3)-O-alpha-glucosyl-PA-maltoheptaose) was hydrolyzed most rapidly, suggesting that it fits the best in the amylo-alpha-1,6-glucosidase active site. It is likely that the active site accommodates 6(2)-O-alpha-glucosyl-maltohexaose and that the interactions of seven glucosyl residues with the active site allow the most rapid hydrolysis of the alpha-1,6-glucosidic linkage of the isomaltosyl moiety.     

Synthesis of 2-Deoxy-glucooligosaccharides through Condensation of 2-Deoxy-D-glucose by Glucoamylase and α-Glucosidase

Glucoamylases from Aspergillus niger and Rhizopus niveus catalyzed condensation of 2-deoxy-D-glucose (dGlc) to yield deoxy-glucooligosaccharides with polymerization degrees of 2–5. The enzymes also gave a small amount of products from 3-deoxy-o-glucose, but no products from 6-deoxy-D-glucose. A. niger α-glucosidase also catalyzed condensation of dGlc, while Torula and Saccharomyces α-glucosidases had low activity. α-l,4-, 1,6-, and 1,3-linked deoxy-glucobioses were isolated and identified as the products of A. niger glucoamylase and A. niger α-glucosidase. In the reaction of the glucoamylase, 1,4- and 1,3-linked saccharides decreased with an increase of 1,6-linked one. A. niger α-glucosidase produced α-1,6-linked disaccharide predominantly during the whole course of the reaction.     

X-ray analysis of 3-O-(6-O-acetyl-2,4-diazido-3-O-benzyl-2,4-dideoxy-alpha-D-gl glucopyranosyl)-1,6-anhydro-2,4-diazido-2,4-dideoxy-beta-D- glucopyranose, a disaccharide having an unusual alpha-D-(1----3) linkage

The crystal structure of 3-O-(6-O-acetyl-2,4-diazido-3-O-benzyl-2,4-dideoxy-alpha-D- glucopyranosyl)-1,6-anhydro- 2,4-diazido-2,4-dideoxy-beta-D-glucopyranose, C21H24N12O7, mol. wt. 556.5, was investigated by X-ray analysis. The disaccharide crystallizes in the triclinic space group P1, with a = 889.3(5), b = 869.6(5), and c = 999.5(6) pm, and alpha = 105.83(4) degrees, beta = 116.22(4) degrees, gamma = 88.42(4) degrees, Z = 1, and rho = 1.394 g.cm-3. Phase determination failed with direct methods, but, as the 1,6-anhydride component of the molecule was already known from a previous structure analysis, the vector-search method could be applied in solving the structure. Diffractometer data were refined to an R value of 0.063 (Rw = 0.080) for 2102 observed reflections. The anhydro-bridged system has a distorted 1C4(D) conformation, in agreement with that of other anhydropyranoses so far investigated. A comparison shows that, for the specific kind of distortion, mainly the anti-reflex effect is responsible, whereas 1,3-diaxial interactions have a minor influence. The nonbridged ring adopts an almost perfect 4C1(D) conformation. The anomeric effect is observed in both of the sugar-ring systems in terms of bond-length shortening. The disaccharide has an alpha-D-Glc-4C1-(1a----3e)-D-Glc-1C4 glycosidic linkage. No previous X-ray investigation of a compound of this type is known. The pyranoid rings are almost perpendicular to each other. The phi, psi angles of the glycosidic linkage are +78.1(5) and -86.0(4) degrees.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of a beta-1,6-clucosidase from Flavobacterium.

An intracellular beta-1,6-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) was produced semiconstitutively by Flavobacterium M64. This enzyme was purified 180-fold by fractionation with ammonium sulfate followed by chromatographies on carboxymethylcellulose, hydroxyapatite and Sephadex G-100. The final preparation appeared homogeneous on disc electrophoresis on polyacrylamide gel. The molecular weight of the enzyme was determined to be ca. 59 000 by Sephadex G-100 gel filtration and sodium dodecylsulfate-polyacrylamide gel electrophoresis. The optimum pH of the enzyme was 5.8 and the optimum temperature was 40 degrees C. The enzyme readily hydrolyzed oligomers with beta-a,6-glucosidic linkages, converting them to glucose. The Km values for gentio-biose, -triose, -tetraose and -pentaose were 2.8, 3.0, 4.2 and 4.6 times 10- minus 4 M, respectively. The rates of their hydrolyses decreased with increase in their chain lengths. The enzyme was concluded to be a beta-1,6-glucosidase from its substrate specificity, production of glucose, transferring ability and inhibition by glucono-delta-lactone. The enzyme activity was inhibited by Hg-2+, Cu-2+, Ag-+, Fe-3+, p-chloromercuribenzoate, N-ethylmaleimide, glucose and trishydroxyaminomethane (Tris) but not by ethylenediaminetetraacetic acid.     

Primary structure of the oligo-1,6-glucosidase of Bacillus cereus ATCC7064 deduced from the nucleotide sequence of the cloned gene

The gene coding for Bacillus cereus ATCC7064 (mesophile) oligo-1,6-glucosidase was cloned within a 2.8-kb SalI-EcoRI fragment of DNA, using the plasmid pUC19 as a vector and Escherichia coli C600 as a host. E. coli C600 bearing the hybrid plasmid pBCE4 accumulated oligo-1,6-glucosidase in the cytoplasm. The cloned enzyme coincided absolutely with B. cereus oligo-1,6-glucosidase in its Mr (65,000), in its electrophoretic behavior on a polyacrylamide gel with or without sodium dodecyl sulfate, in its isoelectric point (4.5), in the temperature dependence of its stability and activity, and in its antigenic determinants. The nucleotide sequence of B. cereus oligo-1,6-glucosidase gene and its flanking regions was determined with both complementary strands of DNA (each 2838 nucleotides). The gene consisted of an open reading frame of 1674 bp commencing with a ATG start codon and followed by a TAA stop codon. The amino acid sequence deduced from the nucleotide sequence predicted a protein of 558 amino acid residues with a Mr of 66,010. The amino acid composition and Mr were comparable with those of B. cereus oligo-1,6-glucosidase. The predicted N-terminal sequence of 10 amino acid residues agreed completely with that of the cloned ligo-1,6-glucosidase. The deduced amino acid sequence of B. cereus oligo-1,6-glucosidase was 72% and 42% similar to those from Bacillus thermoglucosidasius KP1006 (DSM2542, obligate thermophile) oligo-1,6-glucosidase and from Saccharomyces carlsbergensis CB11 alpha-glucosidase, respectively. Predictions of protein secondary structures along with amino acid sequence alignments demonstrated that B. cereus oligo-1,6-glucosidase may take the similar (alpha/beta)8-barrel super-secondary structure, a barrel of eight parallel beta-strands surrounded by eight alpha-helices, in its N-terminal active site domain as S. carlsbergensis alpha-glucosidase and Aspergillus oryzae alpha-amylase.     

枯草芽孢杆菌寡聚—1，6—葡萄糖苷酶基因的克隆及其在大肠杆菌中的表达

本研究用鸟枪法构建了枯草芽孢杆菌（Bacillus subtilis)HB002的基因组文库,经平板法筛选得到了六株能水解合成底物对－硝基苯－α-D－葡萄糖吡喃糖苷的阳性克隆,经鉴定均含克隆了寡聚－1,6－葡萄糖苷酶基因的重组质粒（命名为pHBM001-pHBM006)。选择pHBM003,对其插入片段测序分析,此片段内有一编码561个氨基酸的开放阅读框,该 蛋白质的计算分子量为65.985kD。HB002的寡聚－1,6－葡萄糖苷酶的氨基酸序列与Bacillus sp.和凝结芽孢杆菌（Bacillus coagulans)的寡聚－1,6－葡萄糖苷酶的氨基酸序列一致性分别为81％、67％,相似性分别为89％、79％。从pHBM003中扩增出寡聚－1,6－葡萄糖苷酶基因,克隆到pBV220上,转化大肠杆菌（Escherichia coli)DH5α,得到三个能水解对－硝基苯－α－D－葡萄糖吡喃糖苷的阳性克隆HBM003－1～HBM003－3,将此三个菌株热诱导表达,SDS－PAGE电泳可检测到特异表达的蛋白质,其中HBM003－1、HBM003－2表达的蛋白约66kD,为完整的寡聚－1,6－葡萄糖苷酶,而HBM003－3表达的蛋白质偏小;表达的蛋白质均有寡聚－1,6－葡萄糖苷酶活性。     

Intracellular glycosidases of human colon Bacteroides ovatus B4-11.

Activity of various glycosidases in the intracellular enzyme fraction of Bacteroides ovatus B4-11 was investigated. During 120 h of incubation at 37 degrees C, ca. 30% of the crude hemicellulose was hydrolyzed by an intracellular enzyme fraction of strain B4-11. Xylose was the major sugar released from crude hemicellulose. Glycosidases (alpha-1,6-glucosidase, alpha-1,4-glucosidase, beta-1,4-glucosidase, and beta-1,4-xylosidase) were induced in B. ovatus B4-11 by crude hemicellulose and heteroxylan. When B. ovatus B4-11 was grown on either crude hemicellulose or heteroxylan, the predominant enzyme in the intracellular enzyme fraction was beta-1,4-xylosidase.     

Intracellular glycosidases of human colon Bacteroides ovatus B4-11

Activity of various glycosidases in the intracellular enzyme fraction of Bacteroides ovatus B4-11 was investigated. During 120 h of incubation at 37 degrees C, ca. 30% of the crude hemicellulose was hydrolyzed by an intracellular enzyme fraction of strain B4-11. Xylose was the major sugar released from crude hemicellulose. Glycosidases (alpha-1,6-glucosidase, alpha-1,4-glucosidase, beta-1,4-glucosidase, and beta-1,4-xylosidase) were induced in B. ovatus B4-11 by crude hemicellulose and heteroxylan. When B. ovatus B4-11 was grown on either crude hemicellulose or heteroxylan, the predominant enzyme in the intracellular enzyme fraction was beta-1,4-xylosidase.     

Structural elements in dextran glucosidase responsible for high specificity to long chain substrate

Dextran glucosidase from Streptococcus mutans (SMDG) and Bacillus oligo-1,6-glucosidases, members of glycoside hydrolase family 13 enzymes, have the high sequence similarity. Each of them is specific to alpha-1,6-glucosidic linkage at the non-reducing end of substrate to liberate glucose. The activities toward long isomaltooligosaccharides were different in both enzymes, in which SMDG and oligo-1,6-glucosidase showed high and low activities, respectively. We determined the structural elements essential for high activity toward long-chain substrate. From conformational comparison between SMDG and B. cereus oligo-1,6-glucosidase (three-dimensional structure has been solved), Trp238 and short beta-->alpha loop 4 of SMDG were considered to contribute to the high activity to long-chain substrate. W238A had similar kcat/Km value for isomaltotriose to that for isomaltose, suggesting that the affinity of subsite +2 was decreased by Trp238 replacement. Trp238 mutants as well as the chimeric enzyme having longer beta-->alpha loop 4 of B. subtilis oligo-1,6-glucosidase showed lower preference for long-chain substrates, indicating that both Trp238 and short beta-->alpha loop 4 were important for high activity to long-chain substrates.     

Chemical formation of 4-hydroxy-2,5-dimethyl-3[2H]-furanone from D-fructose 1,6-diphosphate

The selective chemical formation of 4-hydroxy-2,5-dimethyl-3[2H]-furanone (HDF) from D-fructose 1,6-diphosphate in the presence of reduced nicotinamide-adenine-dinucleotides (NAD(P)H) was investigated by means of HPLC-DAD and HPLC-UV-MS/MS. The temperature optimum for HDF formation was 30 degrees C, whereas the pH value (pH 3-10) and chemical nature of the buffer had no significant influence. A linear correlation of reaction time and D-fructose 1,6-diphosphate concentration with the obtained HDF yield was observed. Proteins appeared to have a stabilizing effect. The NAD(P)H were mandatory, even in the presence of protein, implying a non-enzymatic hydride-transfer to an unknown intermediate which finally leads to the selective formation of HDF. The hydride-transfer was confirmed by the application of selectively pro-4R or pro-4S deuterium labeled NADH resulting in each case in the formation of HDF exhibiting a deuterium labeling of approx 30% and employment of [4R,S-(2)H(2)]-NADH led to a deuterium labeling of approx 66%. The incubation of [1-(13)C]-D-fructose 1,6-diphosphate with [4R,S-(2)H(2)]-NADH revealed that the hydride is transferred to C-5 or C-6 of the D-fructose 1,6-diphosphate skeleton. Thus, a chemical HDF formation from D-fructose 1,6-diphosphate under physiological reaction conditions was shown and for the first time to our knowledge a non-enzymatic hydride-transfer from NADH to a carbohydrate structure was demonstrated.     

Crystallization of an anti-2,2,6,6-tetramethyl-1-piperidinyloxy-dinitrophenyl monoclonal antibody Fab fragment with and without bound hapten

The Fab fragment of a monoclonal antibody, AN02, specific for a 2,2,6,6-tetramethyl-1-piperidinyloxy-dinitrophenyl hapten was crystallized both with and without bound hapten. Both crystals formed in phosphate-buffered saline (150 mM-NaCl, 10 mM-Na2PO4, 0.02% (w/v) NaN3 (pH 7.4) at 4 degrees C and diffracted beyond 2.2 A resolution (1A = 0.1 nm). Fab with bound hapten crystallizes in space group P6(1)22 or P6(5)22, with cell dimensions a = b = 73.23 A, c = 373.8 A, alpha = beta = 90 degrees and gamma = 120 degrees. Unoccupied Fab also crystallizes in space group P6(1)22 or P6(5)22 with cell dimensions a = b = 73 A, c = 377 A, alpha = beta = 90 degrees and gamma = 120 degrees.     

Donor substrate specificity of 4-alpha-glucanotransferase of porcine liver glycogen debranching enzyme and complementary action to glycogen phosphorylase on debranching

Glycogen debranching enzyme (GDE) has both 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase activities. Here, we examined 4-alpha-glucanotransferase action of porcine liver GDE on four 6(4)-O-alpha-maltooligosyl-pyridylamino(PA)-maltooctaoses, in the presence or absence of an acceptor, maltohexaose. HPLC analysis of digested fluorogenic branched dextrins revealed that in the presence or absence of acceptor, 6(4)-O-alpha-glucosyl-PA-maltooctaose (B4/81) was liberated from 6(4)-O-alpha-maltopentaosyl-PA-maltooctaose (B4/85), 6(4)-O-alpha-maltotetraosyl-PA-maltooctaose (B4/84) and 6(4)-O-alpha-maltotriosyl-PA-maltooctaose (B4/83), whereas 6(4)-O-alpha-maltosyl-PA-maltooctaose (B4/82) was resistant to the enzyme. The fluorogenic product was further hydrolyzed by amylo-alpha-1,6-glucosidase to PA-maltooctaose (G8PA) and glucose. The ratio of the rates of 4-alpha-glucanotransferase actions on B4/85, B4/84 and B4/83 in the absence of the acceptor was 0.15, 0.42 and 1.00, respectively. The rates increased with increasing amounts of acceptor, changing the ratio of the rates to 0.09, 1.00 and 0.60 (with 0.5 mM maltohexaose) and 0.10, 1.00 and 0.58 (with 1.0 mM maltohexaose), respectively. Donor substrate specificity of GDE 4-alpha-glucanotransferase suggests complementary action of GDE and glycogen phosphorylase on glycogen degradation in the porcine liver. Glycogen phosphorylase degrades the maltooligosaccharide branches of glycogen by phosphorolysis to form maltotetraosyl branches, and phosphorolysis does not proceed further. GDE 4-alpha-glucanotransferase removes a maltotriosyl residue from the maltotetraosyl branch such that the alpha-1,6-linked glucosyl residue is retained.     

Proline residues responsible for thermostability occur with high frequency in the loop regions of an extremely thermostable oligo-1,6-glucosidase from Bacillus thermoglucosidasius KP1006.

The gene encoding for an extremely thermostable oligo-1,6-glucosidase from Bacillus thermoglucosidasius KP1006 (DSM2542, obligate thermophile) was sequenced. The amino acid sequence deduced from the nucleotide sequence of the gene (1686 base pairs) corresponded to a protein of 562 amino acid residues with a Mr of 66,502. Its predicted amino acid composition, Mr, and N-terminal sequence of 12 residues were consistent with those determined for B. thermoglucosidasius oligo-1,6-glucosidase. The deduced sequence of the enzyme was 72% homologous to that of a thermolabile oligo-1,6-glucosidase (558 residues) from Bacillus cereus ATCC7064 (mesophile). B. cereus oligo-1,6-glucosidase contained 19 prolines. Eighteen of these were conserved at the equivalent positions of B. thermoglucosidasius oligo-1,6-glucosidase. This enzyme contained 14 extra prolines besides the conservative prolines. The majority of extra prolines was replaced by polar or charged residues (Glu, Thr, or Lys) in B. cereus oligo-1,6-glucosidase. The extra prolines were responsible for the difference in thermostability between these two enzymes. We suggested that 11 of the extra prolines in B. thermoglucosidasius oligo-1,6-glucosidase occur in beta-turns or in coils within the loops binding adjacent secondary structures.     

Regioselective sulfonylation of 6,1',6'-tri-O-tritylsucrose through dibutylstannylation: synthesis of 4'-O-sulfonyl derivatives of sucrose

3-O-Mesyl-1,6-di-O-trityl-beta-D-fructofuranosyl-(2-->1)-6-O-trityl-alpha-D-glucopyranoside (3) was synthesized via stannylation of 6,1',6'-tri-O-tritylsucrose with dibutyltin oxide in benzene, followed by treatment of the crude product with methanesulfonyl chloride in the presence of triethylamine in dichloromethane at 0 degrees C. A similar treatment of the tri-tritylsucrose in toluene, instead of benzene, yielded 4-O-mesyl-1,6-di-O-trityl-beta-D-fructofuranosyl-(2-->1)-6-O-trityl-alpha-D-glucopyranoside (4) as the major product. The X-ray crystal structure of the corresponding acetyl derivative, 3-O-acetyl-4-O-mesyl-1,6-di-O-trityl-beta-D-fructofuranosyl-(2-->1)-2,3,4-tri-O-acetyl-6-O-trityl-alpha-D-glucopyranoside (5), confirms the position and stereochemistry of the methanesulfonyl group at C-4 of the fructofuranosyl ring.     

Detection of glycogen-debranching system in trophozoites of Entamoeba histolytica

Homogenates of trophozoites of Entamoeba histolytica were shown to bring about the total degradation of glycogen while purified phosphorylase of the same source alone yielded a limit dextrin as end product. An enzyme system capable of debranching the limit dextrin was obtained from the 40,000 g pellet by extraction in aqueous medium, purified by gel filtration on Fractogel TSK HW-55(F), and separated from phosphorylase by chromatography on Blue Sepharose CL-6B and aminobutyl Agarose. The glycogen-debranching system was purified 540-fold to a state of homogeneity by criterion of disc-gel electrophoresis. The purified enzyme was able to degrade glycogen-limit dextrin in the presence of phosphorylase and exhibited activities of both amylo-1,6-glucosidase (EC 3.2.1.33) and 4-alpha-glucanotransferase (EC 2.4.1.25). Although amylo-1,6-glucosidase released glucose from a glycogen-phosphorylase limit dextrin, transferase activity moved single glucose residues from the limit dextrin to 4-nitrophenyl-alpha-glucoside yielding successively 4-nitrophenyl-alpha-maltoside and 4-nitrophenyl-alpha-maltotrioside that could be detected by HPLC. Native glycogen-debranching system exhibited a relative molecular mass of Mr = 180,000 +/- 10% by gel filtration and gel electrophoresis in both denaturing and nondenaturating conditions.     

Crystal structure and molecular conformation of the peptide N-Boc-L-Gly-dehydro-Phe-NHCH3

The peptide N-Boc-L-Gly-dehydro-Phe-NHCH3 was synthesized by the combination of N-Boc-L-Gly-dehydro-Phe azlactone and methylamine. The peptide crystallizes in orthorhombic space group P2(1)2(1)2(1) with a = 5.679(2) A, b = 16.423(9) A, c = 19.198(10) A, V = 1791(2) A3, Z = 4, dm = 1.212(5) Mg m-3, dc = 1.237(1) Mg m-3. The structure was determined by direct methods using SHELXS 86. The structure was refined by full-matrix least squares procedure to an R value of 0.049 for 1509 observed reflections. The molecular dimensions are, in general, in good agreement with the standard values. The bond angle C alpha-C beta-C gamma in the dehydro-Phe residue is 133.6(5) degrees. The peptide backbone torsion angles are theta 1 = -171.4(4) degrees, omega 0 = 178.2(4) degrees, phi 1 = -57.2(6) degrees, psi 1 = 141.2(4) degrees, omega 1 = -174.4(4) degrees, phi 2 = 71.5(6) degrees, psi 2 = 7.2(6) degrees, and omega 2 = -179.8(5) degrees. These values show that the backbone adopts the beta-bend type II conformation. The Boc group has a trans-trans conformation. The side-chain torsion angles in dehydro-Phe are chi 2 = 1.6(9) degrees, chi 2(2, 1) = 0.5(9) degrees, and chi 2(2, 2) = 179.8(6) degrees. The plane of C2 alpha-C2 beta-C2 gamma is rotated with respect to the plane of the phenyl ring at 0.5(6) degrees, which indicates that the atoms of the side chain of the dehydro-Phe residue are essentially coplanar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrolysis of low molecular weight isomaltosaccharides by a p-nitrophenyl-alpha-D-glucopyranoside-hydrolyzing alpha-glucosidase from a thermophile, Bacillus thermoglucosidius KP 1006.

A p-nitrophenyl-alpha-D-glucopyranoside-hydrolyzing alpha-glucosidase of a thermophile, Bacillus thermoglucosidius KP 1006, was purified to an electrophoretically-homogeneous state. Its molecular weight was estimated as 60 000 by gel electrophoresis. The molecular activity (ko) and the Km value at 60 degrees C and pH 6.8 for p-nitrophenyl-alpha-D-glucopyranoside were 233 s-1 and 0.24 mM, respectively. The enzyme cleft the non-reducing terminal alpha-1,6-glucosidic bonds of isomaltose, panose, isomaltotriose, isomaltotetraose, and isomaltopentaose. The ko values were 72.4, 194, 208, 233 and 167 s-1, and the Km values were 3.3, 9.5, 11, 13 and 21 mM, respectively. Each isomaltosaccharide was hydrolyzed to glucose by the cleavage of single glucose units from its nonreducing end. The present study suggests that the enzyme is an oligo-1,6-glucosidase (dextrin 6-alpha-glucanohydrolase, EC 3.2.1.10) and an exo-glucosidase.     

Detection of Glycogen-Debranching System in Trophozoites of Entamoeba histolytica

ABSTRACT. Homogenates of trophozoites of Entamoeba histolytica were shown to bring about the total degradation of glycogen while purified phosphorylase of the same source alone yielded a limit dextrin as end product. An enzyme system capable of debranching the limit dextrin was obtained from the 40,000 g pellet by extraction in aqueous medium, purified by gel filtration on Fractogel TSK HW-55(F), and separated from phosphorylase by chromatography on Blue Sepharose CL-6B and aminobutyl Agarose. The glycogen-debranching system was purified 540-fold to a state of homogeneity by criterion of disc-gel electrophoresis. The purified enzyme was able to degrade glycogen-limit dextrin in the presence of phosphorylase and exhibited activities of both amylo-1,6-glucosidase (EC 3.2.1.33) and 4- α -glucanotransferase (EC 2.4.1.25). Although amylo-1,6-glucosidase released glucose from a glycogen-phosphorylase limit dextrin, transferase activity moved single glucose residues from the limit dextrin to 4-nitrophenyl- α -glucoside yielding successively 4-nitrophenyl- α -maltoside and 4-nitrophenyl- α -maltotrioside that could be detected by HPLC. Native glycogen-debranching system exhibited a relative molecular mass of M_r= 180,000 ± 10% by gel filtration and gel electrophoresis in both denaturing and nondenaturating conditions.     

Synthesis, crystal structure, and molecular conformation of N-Boc-L-Phe-dehydro-Leu-L-Val-OCH3

The peptide N-Boc-L-Phe-dehydro-Leu-L-Val-OCH3 was synthesized by the usual workup procedure and finally by coupling the N-Boc-L-Phe-dehydro-Leu-OH to valine methyl ester. It was crystallized from its solution in methanol-water mixture at 4 degrees C. The crystals belong to the triclinic space group P1 with a = 5.972(5) A, b = 9.455(6) A, c = 13.101(6) A, alpha = 103.00(4) degrees, beta = 97.14(5) degrees, gamma = 102.86(5) degrees, V = 690.8(8) A, Z = 1, dm = 1.179(5) Mg m-3 and dc = 1.177(5) Mg m-3. The structure was determined by direct methods using SHELXS86. It was refined by block-diagonal least-squares procedure to an R value of 0.060 for 1674 observed reflections. The C alpha 2-C beta 2 distance of 1.323(9) A in dehydro-Leu is an appropriate double bond length. The bond angle C alpha-C beta-C gamma in the dehydro-Leu residue is 129.4(8) degrees. The peptide backbone torsion angles are theta 1 = -168.6(6) degrees, omega 0 = 170.0(6) degrees, phi 1 = -44.5(9) degrees, psi 1 = 134.5(6) degrees, omega 1 = 177.3(6) degrees, phi 2 = 54.5(9) degrees, psi 2 = 31.1(10) degrees, omega 2 = 171.7(6) degrees, phi 3 = 51.9(8) degrees, psi T3 = 139.0(6) degrees, theta T = -175.7(6) degrees. These values show that the backbone adopts a beta-turn II conformation. As a result of beta-turn, an intramolecular hydrogen bond is formed between the oxygen of the ith residue and NH of the (i + 3)th residue at a distance of 3.134(6) A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational cycling in beta-phosphoglucomutase catalysis: reorientation of the beta-D-glucose 1,6-(Bis)phosphate intermediate

Activated Lactococcus lactis beta-phosphoglucomutase (betaPGM) catalyzes the conversion of beta-d-glucose 1-phosphate (betaG1P) derived from maltose to beta-d-glucose 6-phosphate (G6P). Activation requires Mg(2+) binding and phosphorylation of the active site residue Asp8. Initial velocity techniques were used to define the steady-state kinetic constants k(cat) = 177 +/- 9 s(-)(1), K(m) = 49 +/- 4 microM for the substrate betaG1P and K(m) = 6.5 +/- 0.7 microM for the activator beta-d-glucose 1,6-bisphosphate (betaG1,6bisP). The observed transient accumulation of [(14)C]betaG1,6bisP (12% at approximately 0.1 s) in the single turnover reaction carried out with excess betaPGM (40 microM) and limiting [(14)C]betaG1P (5 microM) and betaG1,6bisP (5 microM) supported the role of betaG1,6bisP as a reaction intermediate in the conversion of the betaG1P to G6P. Single turnover reactions of [(14)C]betaG1,6bisP with excess betaPGM were carried out to demonstrate that phosphoryl transfer rather than ligand binding is rate-limiting and to show that the betaG1,6bisP binds to the active site in two different orientations (one positioning the C(1)phosphoryl group for reaction with Asp8, and the other orientation positioning the C(6)phosphoryl group for reaction with Asp8) with roughly the same efficiency. Single turnover reactions carried out with betaPGM, [(14)C]betaG1P, and unlabeled betaG1,6bisP demonstrated complete exchange of label to the betaG1,6bisP during the catalytic cycle. Thus, the reorientation of the betaG1,6bisP intermediate that is required to complete the catalytic cycle occurs by diffusion into solvent followed by binding in the opposite orientation. Published X-ray structures of betaG1P suggest that the reorientation and phosphoryl transfer from betaG1,6bisP occur by conformational cycling of the enzyme between the active site open and closed forms via cap domain movement. Last, the equilibrium ratio of betaG1,6bisP to betaG1P plus G6P was examined to evidence a significant stabilization of betaPGM aspartyl phosphate.