Edward J. Hehre

Configurational specificity: Unappreciated key to understanding enzymic reversions and de novo glycosidic bond synthesis: I. Reversal of hydrolysis by α-, β- and glucoamylases with donors of correct anomeric form☆

Abstract Theoretical considerations have led to a new approach for investigating enzymic glycosidic bond formation from free sugars. By use of substrates of defined anomeric form under conditions limiting mutarotation, evidence has been obtained that such condensations require donor substrates of specific configuration. Thus, crystalline glucoamylase from Rhizopus niveus has been found to catalyze the rapid synthesis of maltose and a slower synthesis of isomaltose specifically from β- d -glucopyranose. For the first time, the condensing and hydrolytic activities of this enzyme show a clear correspondence not revealed by past studies of glucosaccharide (mainly isomaltose) formation by prolonged glucoamylase action on mixtures of d -glucose anomers. Crystalline sweet potato β-amylase, likewise, has been found to catalyze the rapid synthesis of maltotetraose specifically from β-maltose, and crystalline hog pancreatic α-amylase the rapid synthesis of maltotetraose specifically from α-maltose. These condensations also confirm for the first time, by synthesis, the specificities of these classic amylases long known from their hydrolytic reactions. A rapid approach to equilibrium was found both in maltose synthesis from β- d -glucopyranose by glucoamylase, and in maltotetraose synthesis from β-maltose by β-amylase. Moreover, essentially the same equilibrium ( K eq = ca . 0.13) was reached by these homologous hemiacetal-to-secondary carbinol condensations. The configurational inversion accompanying both condensations, finally, reveals their mechanism as one of glycosyl transfer. As reversions represent the only class of carbohydrase-catalyzed reactions not previously shown to follow this mechanism, their addition demonstrates the complete generality of the glycosyl-hydrogen interchange model proposed as the paradigm for the action of the carbohydrases.

Stereochemical course of hydrolysis and hydration reactions catalysed by cellobiohydrolases I and II from Trichoderma reesei

Cellobiohydrolase I from Trichoderma reesei catalyzes the hydrolysis of methyl β‐D‐cellotrioside (K m = 48μM, k cat = 0.7 min−1) with release of the β‐cellobiose (retention of configuration). The same enzyme catalyzes the (trans‐hydration of cellobial (K m = 116 μM, K cat = 1.16 min−1) and lactal (K m = 135 μM, k cat = 1.35 min−1), presumably with glycosyi oxo‐carbonium ion mediation. Protonation of the double bond is from the direction opposite that assumed for methyl β‐cellotrioside, but products formed from these prochiral substrates are again of β configuration. Cellobiohydrolase II from the same microrganism hydrolyzes methyl β‐D‐cellotetraoside (K m = 4 μM, k cat = 112 min−1) with inversion of configuration to produce α‐cellobiose. The other reaction product, methyl β‐cellobioside, is in turn partly hydrolysed by Cellobiohydrolase II to form methyl β‐D‐glucoside and D‐glucose, presumably the α‐anomer. Reaction with cellobial is too slow to permit unequivocal determination of product configuration, but clear evidence is obtained that protonation occurs from the si‐direction, again opposite that assumed for protonating glycosidic substrates. These results add substantially to the growing evidence that individual glycosidases create the anomeric configuration of their reaction products by means that are independent of substrate configuration.

The differentiation of serotype A and B dextrans by means of partial acetolysis

Sixteen selected bacterial dextrans were subjected to partial acetolysis, followed by separation and analysis of the disaccharide fragments. Information was obtained bearing on the kinds and proportions of non-1,6-linked units in dextrands and on the chemical basis underlying serotyping of these glucans by type-12 pneumococcus antiserum. The disaccharides found were exclusively α-glucobioses: maltose was recovered from 8 dextrans, kojibiose from 12, and nigerose from all 16 preparations. The latter unexpected finding suggests that dextrans as a class may contain 1,3-linked α- d -glucose as a regular minor structural feature, and that the concept of entirely 1,6-linked dextran may need re-evaluation. For the first time, wide structural differences have been revealed among a number of key dextrans previously found to be identical or closely similar in behavior to periodate oxidation yet different in immunological specificity. Large yields of kojibiose were obtained from those preparations with strong type-12 cross reactivity (serotype A), and none from those devoid of that capacity (serotype B). With all preparations, per cent kojibiose recovered was correlated with extent of reactivity with type-12 pneumococcus antiserum. Among available techniques for studying minor linkages in dextrans, partial acetolysis seems to have an advantage in sensitivity.

The α-amylases as glycosylases, with wider catalytic capacities than envisioned or explained by their representation as hydrolases

Abstract In a study undertaken to illustrate the inadequacy of the familiar concept of carbohydrases as hydrolases, crystalline α-amylases from six different sources, as well as crude salivary amylase, were examined and found to catalyze the synthesis of maltose and maltosaccharides from α- d -glucopyranosyl fluoride, a stereoanalog of α- d -glucopyranose. These syntheses apparently involve initial formation of maltosyl fluoride and higher maltosaccharide 1-fluorides, traces of which were found in digests with certain α-amylases. That the reactions are due to the α-amylases themselves and not to some accompanying enzyme(s) appears certain from the purity and diversity of the preparations; their failure (with one exception) to attack α- or β-maltose; the correspondence of the synthesized products with the known specificity of α-amylases for α-1,4- d -glucosidic linkages (and capacity of different α-amylases to hydrolyze saccharides of different sizes). The “saccharifying” α-amylase of B. sublilis var amylosacchariticus was unique in producing maltosaccharides from both α- and β-maltose (i.e., by α- d -glucosyl transfer). However, the entire group of α-amylases had the capacity to promote α- d -glucosyl transfer from α- d -glucosyl fluoride to C4-carbinol sites, demonstrating for the first time that the catalytic range of α-amylase extends beyond hydrolysis and its reversal. Indeed, all transferred the glucosyl group of α- d -glycosyl fluoride preferentially to C4-carbinols rather than water—a finding neither anticipated nor explained by the representation of α-amylases as hydrolases. The results demonstrate the need to recognize that α-amylases and other hydrolases acting on glycosyl compounds, together with the glycosyl transferases, form a great class of interrelated enzymes whose action is precisely defined as the catalysis of glycosylation (i.e., of glycosyl-hydrogen interchange) and which, therefore, may be designated as “Glycosylases.”

Substrate-induced activation of maltose phosphorylase: Interaction with the anomeric hydroxyl group of α-maltose and α-d-glucose controls the enzyme's glucosyltransferase activity

Maltose phosphorylase, long considered strictly specific for beta-D-glucopyranosyl phosphate (beta-D-glucose 1-P), was found to catalyze the reaction beta-D-glucosyl fluoride + alpha-D-glucose----alpha-maltose + HF, at a rapid rate, V = 11.2 +/- 1.2 mumol/(min.mg), and K = 13.1 +/- 4.4 mM with alpha-D-glucose saturating, at 0 degrees C. This reaction is analogous to the synthesis of maltose from beta-D-glucose 1-P + D-glucose (the reverse of maltose phosphorolysis). In acting upon beta-D-glucosyl fluoride, maltose phosphorylase was found to use alpha-D-glucose as a cosubstrate but not beta-D-glucose or other close analogs (e.g., alpha-D-glucosyl fluoride) lacking an axial 1-OH group. Similarly, the enzyme was shown to use alpha-maltose as a substrate but not beta-maltose or close analogs (e.g., alpha-maltosyl fluoride) lacking an axial 1-OH group. These results indicate that interaction of the axial 1-OH group of the disaccharide donor or sugar acceptor with a particular protein group near the reaction center is required for effective catalysis. This interaction appears to be the means that leads maltose phosphorylase to promote a narrowly defined set of glucosyl transfer reactions with little hydrolysis, in contrast to other glycosylases that catalyze both hydrolytic and nonhydrolytic reactions.

Catalytic versatility of trehalase: Synthesis of α-d-glucopyranosyl α-d-xylopyranoside from β-d-glucosyl fluoride and α-d-xylose

Abstract Trehalase was previously shown (see ref. 5) to hydrolyze α- d -glucosyl fluoride, forming β- d -glucose, and to synthesize α,α-trehalose from β- d -glucosyl fluoride plus α- d -glucose. Present observations further define the enzymes separate cosubstrate requirements in utilizing these nonglycosidic substrates. α- d -Glucopyranose and α- d -xylopyranose were found to be uniquely effective in enabling Trichoderma reesei trehalase to catalyze reactions with β- d -glucosyl fluoride. As little as 0.2m m added α- d -glucose (0.4m m α- d -xylose) substantially increased the rate of enzymically catalyzed release of fluoride from 25m m β- d -glucosyl fluoride at 0°. Digest of β- d -glucosyl fluoride plus α- d -xylose yielded the α,α-trehalose analog, α- d -glucopyranosyl α- d -xylopyranoside, as a transient (i.e., subsequently hydrolyzed) transfer-product. The need for an aldopyranose acceptor having an axial 1-OH group when β- d -glucosyl fluoride is the donor, and for water when α- d -glucosyl fluoride is the substrate, indicates that the catalytic groups of trehalose have the flexibility to catalyze different stereochemical reactions.

Preparation and use of α-maltosyl fluoride as a substrate by beta amylase

Abstract The preparation of pure (amorphous) α-maltosyl fluoride is described. A modification of the procedure of Brauns was used to obtain analytically pure, crystalline hepta- O -acetyl-α-maltosyl fluoride, the structure of which was assigned by 19 F-and 1 H-n.m.r. spectroscopy. α-Maltosyl fluoride was obtained by deacetylating the heptaacetate. It behaved as a single compound on thin-layer and paper chromatography, and was essentially completely hydrolyzed to maltose and hydrogen fluoride by 0.01 M sulfuric acid in 10 min at 100°. Crystalline beta amylase, likewise, catalyzed essentially complete hydrolysis of α-maltosyl fluoride to give maltose and hydrogen fluoride. The rates of hydrolysis catalyzed by beta amylase preparations from sweet potatoes and soybeans acting on a range of concentrations of the substrate produced linear curves for the relationship, 1/ v vs 1/ S ; reaction constants for crystalline, sweet-potato enzyme were K m 3.6 m M and V max ~ 2 μ mol/min/mg. The finding that α-maltosyl fluoride is hydrolyzed 30–60 times faster than maltotriose demonstrates for the first time that beta amylase is capable of effecting hydrolysis at an appreciable rate of a substrate having only two d -glucose residues.

The predominantly nonhydrolytic action of alpha amylases on α-maltosyl fluoride

Abstract Crystalline alpha amylases from a number of sources utilized α-maltosyl fluoride as a glycosyl donor and acceptor at high rates (∼10 to ∼01550 μmol/min/mg of protein, for 30 m M substrate). All enzymes catalyzed conversion of this compound into maltooligosaccharides in preference to causing its hydrolysis. Maltotetraosyl fluoride and maltooligosaccharides of d.p. 3 to 6+ accounted for 75–93% (by weight) of early reaction-products. At a late stage, the yield of maltooligosaccharides was 2–5 times that of maltose, with chains as long as 12 d -glucosyl residues formed by one amylase (from Asp. oryzae ), which utilized α-maltosyl fluoride as a donor and as an acceptor at extremely high rates. These results indicate that alpha amylases have a substantial capacity for binding two molecules of this small substrate in a distinctive way, with the CF glycosylic bond of one and the free C-4 hydroxyl group of the other located in theregion of the enzymes catalytic groups, thereby favoring glycosylation of the suitably positioned acceptor over solvent water. Hydrolysis is assumed to prevail when only a single substrate molecule or segment binds to alpha amylase with a (1→4)-α- d -glucosidic linkage or glycosylic CF bond positioned at the catalytic center. The present demonstration that glycosyl-transfer reactions can be dominantly expressed by alpha amylases, given an appropriate substrate, illustrates the inadequacy of the usual characterization of these enzymes as hydrolases that produce overwhelming hydrolysis of all substrates

A fresh understanding of the stereochemical behavior of glycosylases: Structural distinction of “inverting” (2-MCO-type) versus “retaining” (1-MCO-type) enzymes

Publisher Summary This chapter discusses about the stereochemical behavior of glycosylases. In the traditional view, the catalytic groups of an individual glycosylase act always to invert (or always to retain) substrate configuration, and possibly do so by effecting single (or double) nucleophilic displacements. Glycosyl fluorides of appropriate structure and anomeric configuration serve as substrates for diverse glycosylases, and the catalyzed reactions usually are closely similar in kind and in rate to those promoted with the enzymes best substrates. Yet, on occasion, large departures from this closeness do occur. Thus, alpha-amylases, which usually are thought of as lacking significant glycosyl transferring ability, convert α-maltosyl fluoride to malto-oligsaccharides in great preference to hydrolyzing this substrate, demonstrating that alpha-amylases have a large latent potential for glycosyl transfer. Glucoamylase hydrolyzes α- D -glucosyl fluoride much faster than it cleaves the nonreducing-end D -glucosyl group from maltosaccharides, suggesting that the release of the bulky residual saccharide may perhaps limit the latter substrates rate of hydrolysis. The chapter describes the extraordinary catalytic abilities of glycosylases and presents X-ray findings that support catalytic group versatility and identify the structures controlling stereochemical outcome. It also explains the relation of stereochemical behavior to catalytic mechanism.

Hydrolysis of β-d-glucopyranosyl fluoride to α-d-glucose catalyzed by Aspergillus niger α-d-glucosidase

Abstract Aspergillus niger α- d -glucosidase, crystallized and free of detectable activity for β- d -glucosides, catalyzes the slow hydrolysis of β- d -glucopyranosyl fluoride to form α- d -glucose. Maximal initial rates, V, for the hydrolysis of β- d -glucosyl fluoride, p-nitrophenyl α- d -glucopyranoside, and α- d -glucopyranosyl fluoride are 0.27, 0.75, and 78.5 μmol.min−1.mg−1, respectively, with corresponding V/K constants of 0.0068, 1.44, and 41.3. Independent lines of evidence make clear that the reaction stems from β- d -glucosyl fluoride and not from a contaminating trace of α- d -glucosyl fluoride, and is catalyzed by the α- d -glucosidase and not by an accompanying trace of β- d -glucosidase or glucoamylase. Maltotriose competitively inhibits the hydrolysis, and β- d -glucosyl fluoride in turn competitively inhibits the hydrolysis of p-nitrophenyl α- d -glucopyranoside, indicating that β- d -glucosyl fluoride is bound at the same site as known substrates for the α-glucosidase. Present findings provide new evidence that α-glucosidases are not restricted to α- d -glucosylic substrates or to reactions providing retention of configuration. They strongly support the concept that product configuration in glycosylase-catalyzed reactions is primarily determined by enzyme structures controlling the direction of approach of acceptor molecules to the reaction center rather than by the anomeric configuration of the substrate.