Kinetic Studies on Gluc-amylase

Kitano, Hiromi; Takahashi, Katsutada; Hamauzu, Zen-ichiro; Ono, Shimpei

doi:10.1093/oxfordjournals.jbchem.a128329

Cited by 68 publications

(27 citation statements)

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“…GA catalyzes the hydrolysis of a-1,4 and to a lesser extent a-1,6 glucosidic linkages from the non-reducing end of starch and related glycans to release d-glucose. [46][47][48][49] The active site of GA contains two glutamic acids positioned at positions 179 and 400 of the protein sequence acting as a general acid catalyst and base, respectively. [50,51] The hydrolysis of substrate is accomplished by the donation of a proton from Glu179 to the scissile glycosidic bond and a nucleophilic attack by water on C-1 in the transition-state oxocarbenium ion, catalyzed by Glu400.…”

Section: Wwwchemeurjorgmentioning

confidence: 99%

Chemoselective Capture of Glycans for Analysis on Gold Nanoparticles: Carbohydrate Oxime Tautomers Provide Functional Recognition by Proteins

Thygesen¹,

Sauer²,

Jensen³

2009

Chemistry A European J

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Nanoparticles functionalized with glycans are emerging as powerful solid-phase chemical tools for the study of protein-carbohydrate interactions using nanoscale properties for detection of binding events. Methods or reagents that enable the assembly of glyconanoparticles from unprotected glycans in two consecutive chemoselective steps with meaningful display of the glycan are highly desirable. Here, we describe a novel bifunctional reagent that 1) couples to glycans by oxime formation in solution, 2) aids in purification through a lipophilic trityl tag, and 3) after deprotection then couples to gold nanoparticles through a thiol. NMR studies revealed that these oximes exist as both the open-chain and N-glycosyl oxy-amine tautomers. Glycan-linker conjugates were coupled through displacement of ligands from preformed, citrate-stabilized gold nanoparticles. Recognition of these glycans by proteins was studied with a lectin, concanavalin A (ConA), in an aggregation assay and with a processing enzyme and glucoamylase (GA). We demonstrate that the presence of the N-glycosyl oxy-amines clearly enables functional recognition in sharp contrast to the corresponding reduced oxy-amines. This concept is then realized in a novel reagent, which should facilitate nanoglycobiology by enabling the operationally simple capture of glycans and their biologically meaningful display.

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Section: Wwwchemeurjorgmentioning

confidence: 99%

Chemoselective Capture of Glycans for Analysis on Gold Nanoparticles: Carbohydrate Oxime Tautomers Provide Functional Recognition by Proteins

Thygesen¹,

Sauer²,

Jensen³

2009

Chemistry A European J

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“…5 shows the conformations of active site residues of SBA-maltose, GA-acarbose, and BCX2FXb (2-deoxy-2-fluoro-xylobiose), which were arranged such that they had the same substrate orientation; in each case, the two catalytic residues were positioned above and below the substrate, respectively. Like SBA, GA (glycoside hydrolase family 15) is an inverting exoglycosidase (1, 2) that produces ␤-D-glucose by hydrolyzing ␣-1,4-and ␣-1,6-glucosidic linkage from the non-reducing ends of starch and related oligo-and polysaccharides (44,45 46 and 26% that of the wild-type enzyme, respectively, but the mutant S411A and S411C increased the pH optima when maltose or maltoheptaose was used as the substrate by 0.8 -0.9 pH units; this latter effect was mainly due to the increase in the pK 1 of Glu 400 . Although they did not confirm the loss of the hydrogen bond between Ser 411 and Glu 400 by x-ray crystallographic analysis, the increase in the pK 1 values of S411A or S411C in GA was about the same as that found in SBA mutants (shown in Table II), suggesting that the effect of the disruption of the hydrogen bond between Ser 411 and Glu 400 in S411A or S411C of GA corresponds to that of the disruption of the hydrogen bond between Asn 340 and Glu 380 in both E178Y and N340T in SBA (Fig.…”

Section: ␤-Amylase Mutants With Increased Ph Optimummentioning

confidence: 99%

Structural and Enzymatic Analysis of Soybean β-Amylase Mutants with Increased pH Optimum

Hirata

Adachi

Sekine

et al. 2004

Journal of Biological Chemistry

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Comparison of the architecture around the active site of soybean ␤-amylase and Bacillus cereus ␤-amylase showed that the hydrogen bond networks (Glu 380 -(Lys 295 -Met 51 ) and Glu 380 -Asn 340 -Glu 178 ) in soybean ␤-amylase around the base catalytic residue, Glu 380 , seem to contribute to the lower pH optimum of soybean ␤-amylase. To convert the pH optimum of soybean ␤-amylase (pH 5.4) to that of the bacterial type enzyme (pH 6.7), three mutants of soybean ␤-amylase, M51T, E178Y, and N340T, were constructed such that the hydrogen bond networks were removed by site-directed mutagenesis. The kinetic analysis showed that the pH optimum of all mutants shifted dramatically to a neutral pH (range, from 5.4 to 6.0 -6.6). The K m values of the mutants were almost the same as that of soybean ␤-amylase except in the case of M51T, while the V max values of all mutants were low compared with that of soybean ␤-amylase. The crystal structure analysis of the wild typemaltose and mutant-maltose complexes showed that the direct hydrogen bond between Glu 380 and Asn 340 was completely disrupted in the mutants M51T, E178Y, and N340T. In the case of M51T, the hydrogen bond between Glu 380 and Lys 295 was also disrupted. These results indicated that the reduced pK a value of Glu 380 is stabilized by the hydrogen bond network and is responsible for the lower pH optimum of soybean ␤-amylase compared with that of the bacterial ␤-amylase.

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“…By this method, D was obtained in pure form, but fraction C was contaminated by D. From the commercial preparation (a mixture of A ~ E), D and E were purified in a single isoelectric focusing run. (2), and (3), using the values of the subsite lI:ffinities (A/s) and the intrinsic rate constant (kin') listed in Table III (see text).…”

Section: Purification Of Isozymesmentioning

confidence: 99%

Fractionation of Isozymes and Determination of the Subsite Structure of Glucoamylase fromRhizopus niveus

Tanaka¹,

Fukuchi²,

Ohnishi³

et al. 1983

Agricultural and Biological Chemistry

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Kinetic Studies on Gluc-amylase

Cited by 68 publications

References 0 publications

Chemoselective Capture of Glycans for Analysis on Gold Nanoparticles: Carbohydrate Oxime Tautomers Provide Functional Recognition by Proteins

Chemoselective Capture of Glycans for Analysis on Gold Nanoparticles: Carbohydrate Oxime Tautomers Provide Functional Recognition by Proteins

Structural and Enzymatic Analysis of Soybean β-Amylase Mutants with Increased pH Optimum

Fractionation of Isozymes and Determination of the Subsite Structure of Glucoamylase fromRhizopus niveus

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