Itineraries of enzymatically and non-enzymatically catalyzed substitutions at O-glycopyranosidic bonds (original) (raw)
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Dissecting conformational contributions to glycosidase catalysis and inhibition
Current opinion in structural biology, 2014
Glycoside hydrolases (GHs) are classified into >100 sequencebased families. These enzymes process a wide variety of complex carbohydrates with varying stereochemistry at the anomeric and other ring positions. The shapes that these sugars adopt upon binding to their cognate GHs, and the conformational changes that occur along the catalysis reaction coordinate is termed the conformational itinerary. Efforts to define the conformational itineraries of GHs have focussed upon the critical points of the reaction: substrate-bound (Michaelis), transition state, intermediate (if relevant) and product-bound. Recent approaches to defining conformational itineraries that marry X-ray crystallography of enzymes bound to ligands that mimic the critical points, along with advanced computational methods and kinetic isotope effects are discussed.
Journal of the American Chemical Society, 2007
Using ab initio metadynamics we have computed the conformational free energy landscape of -D-glucopyranose as a function of the puckering coordinates. We show that the correspondence between the free energy and the Stoddard's pseudorotational itinerary for the system is rather poor. The number of free energy minima (9) is smaller than the number of ideal structures (13). Moreover, only six minima correspond to a canonical conformation. The structural features, the electronic properties, and the relative stability of the predicted conformers permit the rationalization of the occurrence of distorted sugar conformations in all the available X-ray structures of -glucoside hydrolase Michaelis complexes. We show that these enzymes recognize the most stable distorted conformers of the isolated substrate and at the same time the ones better prepared for catalysis in terms of bond elongation/shrinking and charge distribution. This suggests that the factors governing the distortions present in these complexes are largely dictated by the intrinsic properties of a single glucose unit.
FEBS Letters, 2002
X-ray crystallography and bioinformatics studies reveal a tendency for the right-handed L L-helix domain architecture to be associated with carbohydrate binding proteins. Here we demonstrate the presence of catalytic L L-helix domains in glycoside hydrolase (GH) families 49, 55 and 87 and provide evidence for their sharing a common evolutionary ancestor with two structurally characterized GH families, numbers 28 and 82. This domain assignment helps assign catalytic residues to each family. Further analysis of domain architecture reveals the association of carbohydrate binding modules with catalytic GH L L-helices, as well as an unexpected pair of L L-helix domains in GH family 55. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.
FEBS Letters, 2004
An in silico survey of all known 3D-structures of glycoside hydrolases that contain a ligand in the À1 subsite is presented. A recurrent crucial positioning of active site residues indicates a common general strategy for electrostatic stabilisation directed to the carbohydrateÕs ring-oxygen at the transition state. This is substantially different depending on whether the en-zymeÕs proton donor is syn or anti positioned versus the substrate. A comprehensive list of enzymes belonging to 42 different families is given and selected examples are described. An implication for an early evolution scenario of glycoside hydrolases is discussed.
ACS Catalysis, 2022
Synthesis methods S2-S4 Synthetic and kinetic schemes (S1 and S2) S5 Key distances for potential and free energy surfaces (Scheme S3) S6 Kinetic parameters for hydrolysis of 1 and 2 by wild-type TmGalA (Table S1) S7 Tables of primers and melting temperatures of variants (Tables S2 and S3) S8-S9 Kinetic parameters for variant enzyme catalyzed reactions with substrates and inhibitors S10-S13 Missing atom types, charges and parameters for 3 and 4 S14-S15 Cartesian coordinates for computed transition states S16-S17 Kinetic plots (Figures S1-S13) S18-S28 Hydrogen-bonding network in wild-type TmGalA complexed with carbasugar (Figure S14) S29 NMR spectra for 6-fluoro-4-methylumbelliferyl -D-melibioside S30-S31 Molecular dynamic simulations S32 Schematic representation of the active site of TmGalA used in the computations S33 References S34
Journal of Organic Chemistry, 2020
Glycoside hydrolases (GHs) catalyze hydrolyses of glycoconjugates in which the enzyme choreographs a series of conformational changes during the catalytic cycle. As a result, some glycoside hydrolase families, including the -amylases (GH13), have their chemical steps concealed kinetically. To address this issue for a GH13 enzyme we made seven cyclohexenylbased carbasugars of -D-glucopyranoside that we show are good covalent inhibitors of a GH13 yeast -glucosidase. The linear free energy relationships between rate constants and pK a of the leaving group is curved upwards, which is indicative of a change in mechanism, with the better leaving groups reacting by a S N 1 mechanism, while reaction rates for the worse leaving groups are limited by a conformational change of the Michaelis complex prior to a rapid S N 2 reaction with the enzymatic nucleophile. Five bicyclo[4.1.0]heptyl-based carbaglucoses were tested with this enzyme, and our results are consistent with pseudo-glycosidic bond cleavage occurs via S N 1 transition states that include non-productive binding of the leaving group to the enzyme. In total we show that the conformationally orthogonal reactions of these two carbasugars reveal mechanistic details hidden by conformational changes that the Michaelis complex of enzyme and natural substrate undergoes that aligns the nucleophile for efficient catalysis.
Molecular basis of substrate specificity in family 1 glycoside hydrolases
IUBMB Life (International Union of Biochemistry and Molecular Biology: Life), 2006
b-glycosidases are active upon a large range of substrates. Besides this, subtle changes in the substrate structure may result in large modifications on the b-glycosidase activity. The characterization of the molecular basis of b-glycosidases substrate preference may contribute to the comprehension of the enzymatic specificity, a fundamental property of biological systems. b-glycosidases specificity for the monosaccharide of the substrate nonreducing end (glycone) is controlled by a hydrogen bond network involving at least 5 active site amino acid residues and 4 substrate hydroxyls. From these residues, a glutamate, which interacts with hydroxyls 4 and 6, seems to be a key element in the determination of the preference for fucosides, glucosides and galactosides. Apart from this, interactions with the hydroxyl 2 are essential to the b-glycosidase activity. The active site residues forming these interactions and the pattern of the hydrogen bond network are conserved among all b-glycosidases. The region of the b-glycosidase active site that interacts with the moiety (called aglycone) which is bound to the glycone is formed by several subsites (1 to 3). However, the majority of the non-covalent interactions with the aglycone is concentrated in the first one, which presents a variable spatial structure and amino acid composition. This structural variability is in accordance with the high diversity of aglycones recognized by b-glycosidases. Hydrophobic interactions and hydrogen bonds are formed with the aglycone, but the manner in which they control the b-glycosidase specificity still remains to be determined.
Carbohydrate Research, 2014
Glycosynthases from more than 16 glycosidase families have been developed for the efficient synthesis of oligosaccharides and glycoconjugates. b-1,3-1,4-Glucan oligo-and polysaccharides with defined sequences can be quantitatively achieved with the glycosynthases derived from Bacillus licheniformis b-1,3-1,4-glucanase. The screening of a nucleophile saturation library of this enzyme yielded the unexpected E134D mutant which has high glycosynthase efficiency (25% higher k cat than the best glycosynthase to date, E134S) but also retains some hydrolase activity (2% relative to the wild-type enzyme). Here, we report the biochemical and structural analyses of this mutant compared to E134S and wild-type enzymes. E134D shows a pH profile of general base catalysis for the glycosynthase activity, with a kinetic pK a (on k cat /K M) assigned to Glu138 of 5.8, whereas the same residue acts as a general acid in the hydrolase activity with the same pK a value. The pK a of Glu138 in the wt enzyme was 7.0, a high value due to the presence of the catalytic nucleophile Glu134 which destabilizes the conjugate base of Glu138. Thus, the pK a of Glu138 drops 1.1 pH units in the mutant relative to the wild-type enzyme meaning that the larger distance between carboxylates in positions 138 and 134 (5.6 Å for wt, 7.0 Å for E134D) and/or a new hydrogen bonding interaction with a third Asp residue (Asp136) in the mutant reduces the effect of the negatively charged Asp134. In consequence, the pK a of Glu138 has a similar pK a value in the E134D mutant than in the other glycosynthase mutants having a neutral residue in position 134. The behavior of the E134D mutant shows that shortening the side chain of the nucleophile, despite maintaining a carboxylate group, confers glycosynthase activity. Therefore E134D is a transitional hydrolase to glycosynthase mutation.