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Glycoside hydrolase enzymes are important for hydrolyzing the β-1,4 glycosidic bond in polysaccharides for deconstruction of carbohydrates. The two-step retaining reaction mechanism of Glycoside Hydrolase Family 7 (GH7) was explored with different sized QM-cluster models built by the Residue Interaction Network ResidUe Selector (RINRUS) software using both the wild-type protein and its E217Q mutant. The first step is the glycosylation, in which the acidic residue 217 donates a proton to the glycosidic oxygen leading to bond cleavage. In the subsequent deglycosylation step, one water molecule migrates into the active site and attacks the anomeric carbon. Residue interaction-based QM-cluster models lead to reliable structural and energetic results for proposed glycoside hydrolase mechanisms. The free energies of activation for glycosylation in the largest QM-cluster models were predicted to be 19.5 and 31.4 kcal mol −1 for the wild-type protein and its E217Q mutant, which agree with experimental trends that mutation of the acidic residue Glu217 to Gln will slow down the reaction; and are higher in free energy than the deglycosylation transition states (13.8 and 25.5 kcal mol −1 for the wild-type protein and its mutant, respectively). For the mutated protein, glycosylation led to a low-energy product. This thermodynamic sink may correspond to the intermediate state which was isolated in the X-ray crystal structure. Hence, the glycosylation is validated to be the rate-limiting step in both the wild-type and mutated enzyme.
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Deprotonated carbohydrate anion fragmentation chemistry: structural evidence from tandem mass spectrometry, infra-red spectroscopy, and theory
We investigate the gas-phase structures and fragmentation chemistry of deprotonated carbohydrate anions using combined tandem mass spectrometry, infrared spectroscopy, regioselective labelling, and theory. Our model system is deprotonated, [lactose-H] − . We computationally characterize the rate-determining barriers to glycosidic bond (C 1 –Z 1 reactions) and cross-ring cleavages, and compare these predictions to our tandem mass spectrometric and infrared spectroscopy data. The glycosidic bond cleavage product data support complex mixtures of anion structures in both the C 1 and Z 1 anion populations. The specific nature of these distributions is predicted to be directly affected by the nature of the anomeric configuration of the precursor anion and the distribution of energies imparted. i.e. , Z 1 anions produced from the β-glucose anomeric form have a differing distribution of product ion structures than do those from the α-glucose anomeric form. The most readily formed Z 1 anions ([1,4-anhydroglucose-H] − structures) are produced from the β-glucose anomers, and do not ring-open and isomerize as the hemiacetal group is no longer present. In contrast the [3,4-anhydroglucose-H] − , Z 1 anion structures, which are most readily produced from α-glucose forms, can ring-open through very low barriers (<25 kJ mol −1 ) to form energetically and entropically favorable aldehyde isomers assigned with a carbonyl stretch at ∼1640 cm −1 . Barriers to interconversion of the pyranose [β-galactose-H] − , C 1 anions to ring-open forms were larger, but still modest (≥51 kJ mol −1 ) consistent with evidence of the presence of both forms in the infrared spectrum. For the cross-ring cleavage 0,2 A 2 anions, ring-opening at the glucose hemiacetal of [lactose-H] − is rate-limiting (>180 (α-), >197 kJ mol −1 (β-anomers)). This finding offers an explanation for the low abundance of these product anions in our tandem mass spectra.
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- Award ID(s):
- 1808394
- PAR ID:
- 10089631
- Date Published:
- Journal Name:
- Physical Chemistry Chemical Physics
- Volume:
- 20
- Issue:
- 44
- ISSN:
- 1463-9076
- Page Range / eLocation ID:
- 27897 to 27909
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
-
Accepted Manuscript
- Journal Article:
- https://doi.org/10.1039/C8CP02620C
