Scope and mechanism of carbohydrase action. Stereocomplementary hydrolytic and glucosyl-transferring actions of glucoamylase and glucodextranase with alpha- and beta-D-glucosyl fluoride.

Kitahata, Sumio; Brewer, C. Fred; Genghof, Dorothy S.; Sawai, Takeshi; Hehre, Edward J.

doi:10.1016/s0021-9258(19)69122-2

Cited by 64 publications

(40 citation statements)

References 47 publications

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“…e study of enzymic glycosylation reactions catalyzed without glycosidic bond cleavage has in recent years emerged as a powerful approach providing a much altered understanding of the catalytic and mechanistic capabilities of glycosylases. That is, through the use of glycosyl fluorides and enolic glycosyl substrates, various well-known glycoside hydrolases and glycosyltransferases have been found to have functionally flexible catalytic groups and the ability to promote glycosylation reactions with different substrates by different mechanisms (Hehre et al, 1977(Hehre et al, , 1979(Hehre et al, , 1980(Hehre et al, , 1982(Hehre et al, , 1986Lehmann & Zieger, 1977; Kitahata et al, 1980;Schlesselmann et al, 1982;Lehmann & Schlesselmann, 1983;Tsumuraya et al, 1984a;Kanda et al, 1986;Kasumi et al, 1986).…”

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confidence: 99%

Catalytic versatility of Bacillus pumilus .beta.-xylosidase: glycosyl transfer and hydrolysis promoted with .alpha.- and .beta.-D-xylosyl fluoride

Kasumi¹,

Tsumuraya²,

Brewer³

et al. 1987

Biochemistry

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Bacillus pumilus beta-xylosidase, an enzyme considered restricted to hydrolyzing a narrow range of beta-D-xylosidic substrates with inversion of configuration, was found to catalyze different stereochemical, essentially irreversible, glycosylation reactions with alpha- and beta-D-xylopyranosyl fluoride. The enzyme promoted the hydrolysis of beta-D-xylopyranosyl fluoride at a high rate, V = 6.25 mumol min-1 mg-1 at 0 degrees C, in a reaction that obeyed Michaelis-Menten kinetics. In contrast, its action upon alpha-D-xylopyranosyl fluoride was slow and characterized by an unusual relation between the rate of fluoride release and the substrate concentration, suggesting the possible need for two substrate molecules to be bound at the active center in order for reaction to occur. Moreover, 1H NMR spectra of a digest of alpha-D-xylosyl fluoride showed the substrate to be specifically converted to alpha-D-xylose by the enzyme. The observed retention of configuration is not consistent with direct hydrolysis by this "inverting" enzyme but is strongly indicative of the occurrence of two successive inverting reactions: xylosyl transfer from alpha-D-xylosyl fluoride to form a beta-D-xylosidic product, followed by hydrolysis of the latter to produce alpha-D-xylose. The transient intermediate product formed enzymically from alpha-D-xylosyl fluoride in the presence of [14C]xylose was isolated and shown by its specific radioactivity and 1H NMR spectrum as well as by methylation and enzymic analyses to be 4-O-beta-D-xylopyranosyl-D-xylopyranose containing one [14C]xylose residue.(ABSTRACT TRUNCATED AT 250 WORDS)

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confidence: 99%

Catalytic versatility of Bacillus pumilus .beta.-xylosidase: glycosyl transfer and hydrolysis promoted with .alpha.- and .beta.-D-xylosyl fluoride

Kasumi¹,

Tsumuraya²,

Brewer³

et al. 1987

Biochemistry

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“…In these cases, mutation of either the catalytic base or the residue responsible for orienting water as a nucleophile , have both proven to be successful strategies. Both methods require that the native glycosidase employ a Hehre resynthesis hydrolysis mechanism , whereby the activated donor is first attacked by an acceptor molecule; only then does hydrolysis of this new glycosidic linkage occur. Glycosynthases interrupt this process such that the addition of the acceptor is completed but active site mutations prevent the subsequent hydrolysis.…”

Section: Glycosynthasesmentioning

confidence: 99%

Advances in Enzymatic Glycoside Synthesis

2016

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A robust platform for facile defined glycan synthesis does not exist. Yet the need for such technology has never been greater as researchers seek to understand the full scope of carbohydrate function, stretching beyond the classical roles of structure and energy storage to encompass highly nuanced cell signaling events. To comprehensively explore and exploit the full diversity of carbohydrate functions, we must first be able to synthesize them in a controlled manner. Toward this goal, traditional chemical syntheses are inefficient while nature's own synthetic enzymes, the glycosyl transferases, can be challenging to express and expensive to employ on scale. Glycoside hydrolases represent a pool of glycan processing enzymes that can be either used in a transglycosylation mode or, better, engineered to function as "glycosynthases," mutant enzymes capable of assembling glycosides. Glycosynthases grant access to valuable glycans that act as functional and structural probes or indeed as inhibitors and therapeutics in their own right. The remodelling of glycosylation patterns in therapeutic proteins via glycoside hydrolases and their mutants is an exciting frontier in both basic research and industrial scale processes.

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“…18 Isolation of the initial transglycosylation product has been described in some reports detailing the "Hehre resynthesis−hydrolysis mechanism." 20,21 Of particular interest to us, this initial transglycosylation reaction closely resembles that of the microscopic reverse reaction, i.e., the standard hydrolysis reaction.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Critically, the initial coupled transglycosylation product has the correct anomeric stereochemistry for it to be a substrate for the GH that made it (Figure B) . Isolation of the initial transglycosylation product has been described in some reports detailing the “Hehre resynthesis–hydrolysis mechanism.” , Of particular interest to us, this initial transglycosylation reaction closely resembles that of the microscopic reverse reaction, i.e., the standard hydrolysis reaction.…”

Section: Introductionmentioning

confidence: 99%

Fundamental Insight into Glycoside Hydrolase-Catalyzed Hydrolysis of the Universal Koshland Substrates–Glycopyranosyl Fluorides

et al. 2021

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We measured a panel of five kinetic isotope effects [KIEs; three secondary deuterium (1-, 2-, and 5-2H) and two primary (1-13C and 5-18O) effects] for the catalyzed hydrolysis of α-d-glucopyranosyl fluoride by two inverting glycoside hydrolases (GHs). The experimental KIEs were compared to average values computed with multiscale QM/MM methods for the hydrolysis of α-d-glucopyranosyl fluoride promoted by an inverting α-glucosidase (Aspergillus niger, GH family 15) to give β-d-glucopyranose, which we explored by the generation of free energy surfaces. We also measured the same KIEs for catalysis of α-d-glucopyranosyl fluoride by the Trichoderma virens GH55 inverting β-glucosidase using our panel of isotopologues; this reaction occurs via the “Hehre resynthesis–hydrolysis mechanism” to give, by two inversions of configuration, the hydrolysis product α-d-glucopyranose. We conclude that the transition states for both enzymatic reactions are essentially identical with fluoride ion departure occurring within a glycoside hydrolase active site that stabilizes pyranosylium ion-like TSs, and with catalysis driven solely by H-bonding assistance from an enzymatic carboxylic acid residue. That is, no assistance is required from the bound nucleophile, which in these two cases is either a water molecule (GH15) or a sugar hydroxyl group (GH55).

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Scope and mechanism of carbohydrase action. Stereocomplementary hydrolytic and glucosyl-transferring actions of glucoamylase and glucodextranase with alpha- and beta-D-glucosyl fluoride.

Cited by 64 publications

References 47 publications

Catalytic versatility of Bacillus pumilus .beta.-xylosidase: glycosyl transfer and hydrolysis promoted with .alpha.- and .beta.-D-xylosyl fluoride

Catalytic versatility of Bacillus pumilus .beta.-xylosidase: glycosyl transfer and hydrolysis promoted with .alpha.- and .beta.-D-xylosyl fluoride

Advances in Enzymatic Glycoside Synthesis

Fundamental Insight into Glycoside Hydrolase-Catalyzed Hydrolysis of the Universal Koshland Substrates–Glycopyranosyl Fluorides

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