A Historical Perspective for the Catalytic Reaction Mechanism of Glycosidase; So As to Bring about Breakthrough in Confusing Situation

Chiba, Seiya

doi:10.1271/bbb.110713

Cited by 15 publications

(13 citation statements)

References 97 publications

(119 reference statements)

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“…One carboxylate protonates the scissile glycosidic oxygen atom and the other coordinates one water molecule, which acts as the nucleophile (55). The ScAraf62A-Ara complex structure showed that the C1 hydroxyl group of the bound L-arabinofuranose molecule assumed a ␤-anomeric conformation and that the distance between the Glu 361 -O⑀1 and the Ara-C1 atoms was 3.8 Å.…”

Section: Catalytic Mechanisms and Substrate Specificities Of Gh62 Enzmentioning

confidence: 99%

Crystal Structure and Characterization of the Glycoside Hydrolase Family 62 α-l-Arabinofuranosidase from Streptomyces coelicolor

Maehara

Fujimoto

Ichinose

et al. 2014

Journal of Biological Chemistry

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Background: Glycoside hydrolase family 62 ␣-L-arabinofuranosidases specifically release L-arabinose from arabinoxylan. Results: The crystal structure of glycoside hydrolase family 62 ␣-L-arabinofuranosidase from Streptomyces coelicolor was determined. Conclusion: L-Arabinose and xylohexaose complexed structures revealed the mechanism of substrate specificity of this enzyme. Significance: Efficient catalysis by glycoside hydrolase family 62 ␣-L-arabinofuranosidase requires its binding to terminal xylose sugars at the substrate-binding cleft.

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Section: Catalytic Mechanisms and Substrate Specificities Of Gh62 Enzmentioning

confidence: 99%

Crystal Structure and Characterization of the Glycoside Hydrolase Family 62 α-l-Arabinofuranosidase from Streptomyces coelicolor

Maehara

Fujimoto

Ichinose

et al. 2014

Journal of Biological Chemistry

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“…8 The retaining mechanism has been proposed for α-amylase, 9 maintaining the initial conformation on the anomeric carbon. …”

Section: ■ Introductionmentioning

confidence: 99%

Establishing the Catalytic Mechanism of Human Pancreatic α-Amylase with QM/MM Methods

Pinto

Brás

Perez

et al. 2015

J. Chem. Theory Comput.

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In this work, we studied the catalytic mechanism of human pancreatic α-amylase (HPA). Our goal was to determine the catalytic mechanism of HPA with atomic detail using computational methods. We demonstrated that the HPA catalytic mechanism consists of two steps, the first of which (glycosylation step) involves breaking the glycosidic bond to culminate in the formation of a covalent intermediate. The second (deglycosylation step) consists of the addition of a water molecule to release the enzyme/substrate covalent intermediate, completing the hydrolysis of the sugar. The active site was very open to the solvent. Our mechanism basically differs from the previously proposed mechanism by having two water molecules instead of only one near the active site that participate in the mechanism. We also demonstrate the relevant role of the three catalytic amino acids, two aspartate residues and a glutamate (D197, E233, and D300), during catalysis. It was also shown that the rate limiting step was glycosylation, and its activation energy was in agreement with experimental values obtained for HPA. The experimental activation energy was 14.4 kcal mol(-1), and the activation energy obtained computationally was 15.1 kcal mol(-1).

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“…Two models for the catalytic mechanism of β-glycoside hydrolases have been proposed: (i) a double displacement pathway first proposed by Koshland, essentially proceeding as an S N 2 reaction (11,12) and (ii) an oxocarbenium ion intermediate pathway originally proposed by Phillips (13), occurring as an S N 1 reaction (Fig. S1) (14,15). The Koshland mechanism suggests a glycosylation step, in which a covalent glycosyl-enzyme intermediate bearing inverted stereochemistry at the anomeric C1 is formed, followed by the deglycosylation step, in which the latter is hydrolyzed by an incoming H 2 O activated by the first Glu residue and switched its role to act as a general base.…”

mentioning

confidence: 99%

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

Wan

Parks

Hanson

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pK a values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD = pH + 0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pK a values of the catalytic Glu residues in both the apo-and substrate-bound states of the enzyme. The calculated pK a of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. These findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.glycoside hydrolase | protonation state | macromolecular neutron crystallography | xylanase | molecular simulations

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A Historical Perspective for the Catalytic Reaction Mechanism of Glycosidase; So As to Bring about Breakthrough in Confusing Situation

Cited by 15 publications

References 97 publications

Crystal Structure and Characterization of the Glycoside Hydrolase Family 62 α-l-Arabinofuranosidase from Streptomyces coelicolor

Crystal Structure and Characterization of the Glycoside Hydrolase Family 62 α-l-Arabinofuranosidase from Streptomyces coelicolor

Establishing the Catalytic Mechanism of Human Pancreatic α-Amylase with QM/MM Methods

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

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