Glucosidase-Catalyzed Hydrolysis of α-<scp>d</scp>-Glucopyranosyl Pyridinium Salts:  Kinetic Evidence for Nucleophilic Involvement at the Glucosidation Transition State

Huang, Xicai; Tanaka, and Kelly S. E.; Bennet, Andrew J.

doi:10.1021/ja963733l

Cited by 60 publications

(42 citation statements)

References 43 publications

(91 reference statements)

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“…Evidence supporting an S N 2-like mechanism involving enzymatic nucleophilic catalysis comes from the observation of normal secondary a deuterium KIEs for both transition states (indicating rehybridization of C1 from sp 3 to sp 2 ), 9 as well as the observation of primary 13 C KIEs w 1.01 (indicating reaction coordinate motion contributions from both the nucleophile and the leaving group) consistent with S N 2 pathways. 10,11 Additionally, the covalent glycosyl-enzyme intermediates from representatives of multiple families have been isolated and characterized using mass spectrometry and X-ray crystallography. This includes the characterization of the intermediate of lysozyme, a result that contradicts the classical textbook mechanism involving an oxocarbenium ion intermediate.…”

Section: Glycosidases and Transglycosidasesmentioning

confidence: 99%

Mechanistic analogies amongst carbohydrate modifying enzymes

Lairson

Withers

2004

Chem. Commun.

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Carbohydrates are known to play essential roles in a myriad of biological processes. The enormous complexity of the various oligosaccharide structures found in biology is derived from a rational orchestration of the enzymatic formation and breakdown of glycosidic linkages achieved by glycosyltransferases, glycosidases and phosphorylases. A detailed understanding of the chemical mechanisms by which these classes of enzymes function not only provides a rational basis for their engineering and application in both the development and synthesis of new classes of therapeutic agents, but also provides insight into the role of convergence in the natural evolution of enzyme function.

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Section: Glycosidases and Transglycosidasesmentioning

confidence: 99%

Mechanistic analogies amongst carbohydrate modifying enzymes

Lairson

Withers

2004

Chem. Commun.

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“…This canonical double-displacement mechanism has been challenged by proponents of the oxocarbenium ion intermediate mechanism, in which the general acid-assisted departure of an aglycon leaving group leads to a short-lived, planar carbocation on C1 of the glycon, which is then hydrated by an incoming H 2 O with an estimated rate constant of 10 12 s −1 (16). Each mechanism draws support from different types of experimental and computational evidence (17)(18)(19)(20)(21)(22), with the double-displacement mechanism being generally accepted.…”

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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“…Subsequent use of the Furukawa modification of the Simmons-Smith reaction gave the two cyclopropyl isomers in a total yield of 88%. 33 Of note, the ratio of the D-galacto (18) and L-altro (19) diastereomers formed in this reaction was unpredictable, that is, it varied between 2.5 : 1.0 and 1.0 : 1.5. The absolute stereochemistry for the two diastereomers was assigned based on observed NOE contacts between one of the H-7 protons and either H-3 or H-4 in the 1D NOE difference spectra (Fig.…”

Section: Resultsmentioning

confidence: 95%

“…While no single compound effectively inhibits all galactosidases 12-14 most inhibitors possess structural features that mimic certain facets of the galactopyranosylium ion (1), a high-energy intermediate formed during the acid-catalysed hydrolysis of galactosides in aqueous solution. [15][16][17] Regardless of whether the enzymecatalysed glycosylation and deglycosylation steps are dissociative (D N * A N ) 18, 19 or "exploded" associative (A N D N ) 19,20 reactions, it is clear that there is a sizable degree of cationic character on the sugar moiety at the critical enzymatic TSs, 21, 22 which therefore must bear some resemblance to the galactopyranosylium ion. Thus, 1deoxy-galacto-nojirimycin (2), 23,24 galacto-isofagomine (3) 12, 25 and galacto-validamine (4) 26 mimic charge development at the TS by incorporating a basic nitrogen atom in place of O-5, C-1, and O-1, respectively (Fig.…”

Section: Introductionmentioning

confidence: 99%

A potent bicyclic inhibitor of a family 27 α-galactosidase

Wang

Bennet

2007

Org. Biomol. Chem.

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Two isomeric bicyclo[4.1.0]heptane analogues of the glycosidase inhibitor galacto-validamine, (1R*,2S,3S,4S,5S,6S*)-5-amino-1-(hydroxymethyl)bicyclo[4.1.0]heptane-2,3,4-triol, have been synthesized in 13 steps from 2,3,4,6-tetra-O-benzyl-D-galactose. The inhibitory activities of the two conformationally restricted amines, and their corresponding acetamides, were measured against commercial alpha-galactosidase enzymes from coffee bean and E. coli. The activity of the glycosyl hydrolase family GH27 enzyme (coffee bean) was competitively inhibited by the 1R,6S-amine (7), a binding interaction that was characterized by a K(i) value of 0.541 microM. The GH36 E. coli alpha-galactosidase exhibited a much weaker binding interaction with the 1R,6S-amine (IC(50)= 80 microM). The diastereomeric 1S,6R-amine (9) bound weakly to both galactosidases, (coffee bean, IC(50)= 286 microM) and (E. coli, IC(50)= 2.46 mM).

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Glucosidase-Catalyzed Hydrolysis of α-d-Glucopyranosyl Pyridinium Salts: Kinetic Evidence for Nucleophilic Involvement at the Glucosidation Transition State

Cited by 60 publications

References 43 publications

Mechanistic analogies amongst carbohydrate modifying enzymes

Mechanistic analogies amongst carbohydrate modifying enzymes

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

A potent bicyclic inhibitor of a family 27 α-galactosidase

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