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Query: EC:3.2.1.21 (beta-glucosidase)
 3,280 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The role of noncovalent interactions in the catalytic mechanism of the Agrobacterium faecalis beta-glucosidase was investigated by steady-state and pre-steady state kinetic analysis of the hydrolysis of a series of monosubstituted aryl glycosides, in which the hydroxyl groups on the glycone were substituted by hydrogen or fluorine. Contributions of each hydroxyl group to binding of these substrates at the ground state are relatively weak (interaction energies of 3.3 kJ/mol or smaller) but are much greater at the two transition states (glycosylation and deglycosylation). The strongest transition state interactions were at the 2 position (at least 18 and 22 kJ/mol for glycosylation and deglycosylation, respectively) with the interactions at the 3 and 6 positions contributing at least another 9 kJ/mol of binding energy at both transition states. The interaction at the 4 position is less crucial to transition state binding but important for stabilization of the glycosyl-enzyme intermediate. Comparison of observed rates with those for spontaneous hydrolysis of the same substrates provides evidence for oxocarbenium ion character at both transition states, that for deglycosylation apparently having the greater positive charge development at the anomeric center.
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PMID:Mechanism of Agrobacterium beta-glucosidase: kinetic analysis of the role of noncovalent enzyme/substrate interactions. 851 77

D-Glucal and a series of substituted derivatives have been tested as substrates, inhibitors and inactivators of the Agrobacterium faecalis beta-glucosidase in order to probe structure/function relationships in this enzyme. D-Glucal is shown to be a substrate (kcat = 2.3 min-1, Km = 0.85 mM) undergoing hydration with stereospecific protonation from the alpha-face to yield 2-deoxy-beta-D-glucose. 1-Methyl-D-glucal surprisingly serves as only a poor substrate (kcat = 0.056 min-1, Km = 57 mM), also undergoing protonation from the alpha-face. 2-Fluoro-D-glucal, however is completely inert, as a result of inductive destabilisation of the oxocarbenium ion-like transition state for protonation, and functions only as a relatively weak (Ki = 24 mM) inhibitor. Similar behaviour was seen with almond beta-glucosidase and yeast alpha-glucosidase and for the interaction of 2-fluoro-D-galactal with Escherichia coli beta-galactosidase. A series of of alpha, beta-unsaturated glucal derivatives was also synthesised and tested as potential substrates, inhibitors or inactivators of A. faecalis beta-glucosidase. Of these only 1-nitro-D-glucal functioned as a time dependent, irreversible inactivator (ki = 0.011 min-1, Ki = 5.5 mM), presumably acting as a Michael acceptor. Electrospray mass spectrometric analysis revealed multiple labeling of the enzyme by this inactivator, lessening its usefulness as an affinity label. Less reactive Michael acceptor glycals which might have been more specific (1-cyano-, 2-cyano-, 1-carboxylic acid, 1-carboxylic acid methyl ester) unfortunately did not function as inactivators or substrates, only as relatively weak reversible inhibitors (Ki = 3-96 mM).
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PMID:Substituted glycals as probes of glycosidase mechanisms. 900 77

Glycosynthases are nucleophile mutants of retaining glycosidases that catalyze the glycosylation of sugar acceptors using glycosyl fluoride donors, thereby synthesizing oligosaccharides. The 'original' glycosynthase, derived from Agrobacterium sp. beta-glucosidase (Abg) by mutating the nucleophile glutamate to alanine (E358A), synthesizes oligosaccharides in yields exceeding 90% [Mackenzie, L.F., Wang, Q., Warren, R.A.J. and Withers, S.G. (1998) J. Am. Chem. Soc. 120, 5583-5584]. This mutant has now been re-cloned with a His(6)-tag into a pET-29b(+) vector, allowing gram scale production and single step chromatographic purification. A dramatic, 24-fold, improvement in synthetic rates has also been achieved by substituting the nucleophile with serine, resulting in improved product yields, reduced reaction times and an enhanced synthetic repertoire. Thus poor acceptors for Abg E358A, such as PNP-GlcNAc, are successfully glycosylated by E358S, allowing the synthesis of PNP-beta-LacNAc. The increased glycosylation activity of Abg E358S likely originates from a stabilizing interaction between the Ser hydroxyl group and the departing anomeric fluorine of the alpha-glycosyl fluoride.
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PMID:The E358S mutant of Agrobacterium sp. beta-glucosidase is a greatly improved glycosynthase. 1064 8