THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 254, No. 13, Issue of July 10, pp. 5942-5950, 1979 Printed m U.S.A.

Scope and Mechanism of Catibohydrase Action HYDROLYTIC AND NONHYDROLYTIC ACTIONS OF ,8-AMYLASE ON a- AND /3-MALTOSYL FLUORIDE*

(Received for publication, November 9, 1978)

Edward J. Hehre, Curtis F. Brewer,+ and Dorothy S. Genghof

From the Department of Microbiology and Immunology and Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461

A unique demonstration is presented of the capacity of a carbohydrase to utilize both anomeric forms of a substrate. Crystalline sweet potato P-amylase has been found to act upon a- and P-maltosyl fluoride, forming p-maltose and hydrogen fluoride in each case. The re- action with P-maltosyl fluoride is distinguished by an unusual relationship of rate to substrate concentration, indicative of the need for 2 molecules of substrate to be bound to enzyme in order for reaction to occur. In addition, certain compounds with an a-o-glucopyrano- syl terminal, e.g. methyl P-maltoside and p-nitrophenyl a-n-glucoside, greatly enhance the rate of utilization of low levels of /3-maltosyl fluoride by the enzyme. The reaction pathway that accounts for these results in- volves two steps: maltosyl transfer from /3-maltosyl fluoride to the C-4 hydroxyl of a second molecule of substrate (or alternate acceptor) to form an a-1,4-linked higher saccharide; then, rapid hydrolysis of the latter to give p-maltose. The first step is similar to the earlier reported condensation of p-maltose to maltotetraose catalyzed by /I-amylase (Hehre, E. J., Okada, G., and Genghof, D. S. (1969) Arch. Biochem. Biophys. 135, 75- 89); the second step is as for the hydrolysis of a-1,4 linkages of amylaceous substrates, and accounts also for the hydrolysis of a-maltosyl fluoride to /3-maltose.

P-Amylase thus emerges as the catalyst of two types of reactions: hydrolysis of a-maltosyl substrates to give p-maltose, and maltosyl transfer from P-maltosyl sub- strates to carbohydrates to form new a-1,4-glycosidic linkages. To account for these complementary reaction patterns, a mechanism is proposed for /I-amylase that involves reversible general acid-base catalysis by an imidazole and a carboxyl residue at the active site, possibly by way of a concerted mechanism. Each fimc- tional group is viewed as having a dual role, reversed for the two types of reactions. Special significance at- taches to the catalysis of maltosyl transfer reactions from /3-maltosyl fluoride as all inverting glycanases, heretofore, have been considered hydrolases that cat- alyze only reactions involving water as a reactant. Present results strongly support the concept that gly- coside hydrolases and glycosyl transferases are inter- related catalysts of glycosylation, ie. glycosylases.

* This study was supported, in part, by a grant from the Corn Refiners Association, Inc. (to E. J. H.), by Public Health Service Research Grant GM-25478 from the National Institute of General Medical Sciences (to E. J. H.) and by Grant 5-KO4CAOOW from the National Cancer Institute (to C. F. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

f Recipient of Research Career Development Award 5-K04- CA00184 from the National Cancer Institute.

P-Amylase, 1,4-a-D-glucano maltohydrolase [EC 3.2.1.21 (l), has long been understood to catalyze a single type of reaction: the hydrolysis of 1,4-a-n-glycosidic linkages of starch, glyco- gen, and maltosaccharides in such a way that successive maltosyl residues are cleaved from the chain ends and released as p-maltose (2-10). In particular, past studies provide no indication that /3-amylase can catalyze any reaction other than those which involve water as a reactant and which lead to inversion of configuration. These limits of P-amylase action offered a unique opportunity to test the effectiveness of a new method for studying the activities of carbohydrates, based on the concept that these enzymes are catalysts of glycosyl- proton interchange (11). This method, consisting of the study of glycosylation reactions that occur without glycosidic bond cleavage, has recently led to a new level of understanding of the catalytic capabilities of several well known carbohydrases (12-19). By use of this approach, we have now obtained evidence that /?-amylase has a transglycosylative activity in addition to the hydrolytic activity hitherto represented as its sole catalytic capacity. For the first time, also, the enzyme has been shown to catalyze a hydrolytic conversion resulting in retention of configuration, which can be accounted for by the sequential catalysis of a transglycosylation and a hydrolytic reaction.

In 1969, crystallized sweet potato P-amylase was shown in our laboratory (19) to catalyze the synthesis of maltotetraose (maltosyl-a-1,4-maltose) from p-maltose. This stereospecific condensation, demonstrating the principle of microscopic re- versibility with the hydrolytic reaction (10, 19-22), provided a fresh perspective for viewing the catalytic potentialities of /3-amylase. That is, the synthesis from p-maltose showed the enzyme to have the capacity to cleave a glycosylic bond that is not part of a glycosidic linkage, to utilize a ,&maltosyl compound as a substrate, and to transfer a maltosyl residue to the free C-4 hydroxyl group of a maltose molecule with inversion of configuration from p- to a-. These capacities suggested the possibility that ,6-amylase might cleave other glycosylic bonds that are not part of a glycosidic linkage, utilize other P-maltosyl compounds as substrates, and catalyze other maltosyl transfer reactions with inversion of configura- tion from p- to a-, but without releasing water as a product.

The first of these possibilities has recently been realized. We have found that ,&amylases not only catalyze hydrolysis of the C-F glycosylic bond of a-maltosyl fluoride but do so 30 to 60 times faster than they hydrolyze the a-1,4-n-glycosidic bonds of maltotriose (16). The present work examines the remaining two possibilities, using ,8-maltosyl fluoride as the probe. We envisioned that this stereoanalog of ,&maltose would have characteristics enabling it to be a far better substrate for /I-amylase than /?-maltose, just as a-maltosyl fluoride is a far better substrate (for a-amylase) than a-mal- tose (11, 17). Replacement of the anomeric hydroxyl group of

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Hydrolytic and Nonhydrolytic Actions of ,B-Amylase 5943

,&maltose by a fluorine atom should, because of the similarity in the OH and fluorine atomic dimensions, allow P-maltosyl fluoride to bind to ,&amylase in the same manner as ,&maltose.

The great susceptibility of the glycosylic C-F bond to cleav- age, plus the high heat of hydration, tight binding of water, and strong hydrogen bonding ability of the liberated fluoride anion (23), should ensure the almost complete irreversibility

of enzymic reactions involving fluoride release from p-malto- syl fluoride.

P-Maltosyl fluoride is, in fact, an active substrate for /3- amylase. Its conversion to p-maltose and hydrogen fluoride by the enzyme, and the kinetic distinction between this con- version and the hydrolysis of a-maltosyl fluoride which the enzyme also catalyzes (16) have been reported in a preliminary communication (18). Apparently, this is the fist known in- stance of the utilization of both (Y and p anomeric forms of a compound with cleavage of their anomerically distinct glyco- sylic bonds, by any carbohydrase.

The present paper describes the chemical synthesis and purification of /?-maltosyl fluoride and the results of experi- ments carried out to elucidate the mode of action of ,&amylase on the compound. As witl be shown, this mode is stereospe- cifically complementary to that represented by the hydrolysis of a-maltosyl fluoride and of a-glucans of the starch-glycogen class by the enzyme. The bearing of these findings on the catalytic mechanism of /?-amylase, and on the thesis that glycoside hydrolases and glycosyl transferases form a single, large class of glycosylases, is discussed.

EXPERIMENTAL PROCEDURES

Methods

General-Thin layer chromatography (tic) was carried out using Silica Gel G (Brinkmann Instruments, Westbury, N. Y.) with ethyl ether, 0.5% water (Mallinckrodt) as solvent for acetylated compounds and absolute ethanol:ethyl acetate (2:5) for nonacetylated com- pounds. Spots were visualized by the sulfuric acid-char method. Paper chromatograms were developed with 1-butanol/pyridine/water, 6:4:3 (ascending), and stained by a silver nitrate dipping technique (24) with papers hung in air for 10 min following application of the alkaline reagent.

Concentrations were carried out under reduced pressure in rotary evaporators, at or below 35°C. Drying was done in a vacuum oven at or below 35°C. Crystallinity was judged by the use of a polarizing microscope equipped with a fist order (red) retardation plate. Melting points were determined on a Mel-Temp block (Laboratory Devices, Cambridge, Mass.) and are uncorrected. Optical rotations were meas- ured with a Rudolph and Sons model 70 polarimeter and 2-dm tubes or, where indicated, with a Perkin-Elmer model 141 photoelectric polarimeter and l- or O.l-dm cells. Total carbohydrate was determined by the phenolsulfuric acid method (25), standardized concurrently with n-glucose. Elemental analyses were performed by Galbraith Laboratories Inc., Knoxville, Tenn.

Fluoride Determination-Measurements of free fluoride ion, in the presence or absence of maltosyl fluoride, were made with the aid of a combination fluoride electrode, model 96-09, and specific ion meter, model 407A (Orion Research, Inc., Cambridge, Mass.). Sodium fluoride solutions of known concentration were used as concurrent standards. Standards and test samples were fist diluted with an equal volume of a solution comprising 1 M sodium acetate buffer (pH 5.2), 1 M sodium chloride, and 0.4% 1,4cyclohexane bis(dinitrilotetraacetic acid) monohydrate and then poured into 5-ml polyethylene beakers. Meter readings were recorded 45-s after immersion of the electrode in the solutions under test.

Nuclear Magnetic Resonance Spectra-‘H, ‘“C, and lYF NMR spectra at 23.5 kG were obtained using a Jeol PFT spectrometer interfaced with a Nicolet 1000 series computer. The instrument was operated in the pulse Fourier transform mode. NMR spectra of hepta- 0acetyl /l-maltosyl fluoride were recorded using deuterobenzene as solvent. lYF chemical shift measurements were made with respect to trichlorofluoromethane which was used as an internal standard. ‘H NMR spectra of P-maltosyl fluoride and maltose were recorded in 0.2 M acetate-d,/deuterium oxide buffer, pD 5.7; chemical shift measure-

ments were made with respect to 3-(trimethylsilyl)propanesulfonic acid sodium salt (Thompson-Packard, Little Falls, N. J.) as an internal standard.

Materials

Crystalline sweet potato /3-amylase preparations (-1000 units/mg of protein) of two types were used. One was a commercial product (Sigma, type I-B); the second, which had been further fractionated to remove accompanying a-glucosidase (26), was the gift of Drs. W. J. Whelan and J. J. Marshall. Immediately before use, the enzymes were separated from the ammonium sulfate suspension fluids by centrifu- gation and dissolved in ice-cold buffer to provide solutions of desired composition.

Purified cY-maltosyl fluoride was prepared as recently described (16). p-Nitrophenyl a-n-glucopyranoside, phenyl and p-nitrophenyl P-n-glucopyranoside, sorbitol, and mannitol were reagent grade prod- ucts (Sigma). Glucose and maltose monohydrate were specially puri- fied laboratory preparations; lOO+g samples gave a single spot on chromatography. Deuterium oxide (99.7 atom ‘% D) was purchased from Merck, Sharpe and Dohme, Ltd., Canada; acetic-d:3 acid sodium salt (99+ atom % D) from Aldrich Chemical Co., Milwaukee, Wise.; methanol-& (99 atom o/o D) from Prochem, Ltd., Croyden, England.

Hepta-0.acetyl a-Maltosyl Bromide-Synthesis was by the pro- cedure of Brauns (27). A solution containing 20 g of recrystallized /l- maltose octaacetate (m.p., 160-161’C and [o]?? + 63.5” (c 3, chloro- form)), 70 ml glacial acetic acid, and 40 ml of 31% hydrogen bromide (Eastman Kodak) was kept at 0°C for 30 min and then shaken with a mixture of ice water and chloroform. The organic phase was thor- oughly washed with water, dehydrated with sodium sulfate, then concentrated to a thick syrup and dried in a vacuum oven (35°C). The product (average yield, 19 g) was an amorphous white powder, [o]E50 + 169” (c 2, chloroform); literature value (27), [a]?’ + 180.1” (chloroform). Efforts to remove a minor impurity evident on tic were not successful.

Hepta-0-acetyl P-Maltosyl Fluoride-This was synthesized by a procedure analogous to that reported (28, 29) for tetra-O-acetyl D-D- glucopyranosyl fluoride. In a typical run, 10 g of hepta-0-acetyl a- maltosyl bromide in 55 ml of acetonitrile was shaken mechanically with 5 g of pulverized silver fluoride (Alfa Division, Ventron Corp., Danvers, Mass.) for 4 h at 25°C. Following filtration and solvent evaporation under vacuum (35’(Z), -20 ml of a cloudy syrup was obtained. This was taken up in 65 ml of chloroform, thoroughly washed with water, and dried with sodium sulfate. Concentration now yielded a clear, thick syrup. Treatment with ethyl ether (120 ml) gave a clear solution which, within 10 min at 25”C, became a heavy suspension of crystals. These appeared as sturdy, birefringent needles on polarization microscopy. (In other runs, a mixture of amorphous particles and crystals was found at this stage.) The recovered product (5.1 g; m.p., 126-129°C) showed on tic (ether) a major spot (RF = 0.7) accompanied by a minor spot (RF - 0.55)). Purification was carried out as follows.

Samples (1 g) of the crude acetate were individually chromato- graphed on fresh columns (2.5 x 30 cm) of dry Silica Gel 60 (70 to 230 mesh, E. Merck, Darmstadt, Germany), developed with ethyl ether (16). Fractions showing a single spot (RF - 0.7) on tic were pooled and dried; those showing a small amount of impurity were dried and rechromatographed. The purified (single-spot) material from 8.3 g of the impure substance weighed 2.8 g. This was dissolved in a small volume of chloroform, and the solution was evaporated under vacuum (35°C) to near dryness (bubbling stage); 120 ml of ethyl ether then was added, with shaking, to produce a water clear solution. Crystal- lization began within a few minutes and was allowed to proceed for 24 h at 4’C. The final product (2.64 g) had values of m.p. 130-132OC and [a]?’ + 80.1’ (c 0.8, chloroform).

CZH:IROI~F Found: C 48.91, H 5.52, F 2.90

Calculated: C 48.92, H 5.48, F 2.97

/I-Maltosyl Fluoride-Purified P-maltosyl fluoride heptaacetate (255 mg) in 0.75 ml of chloroform was treated with 5 volumes of ice- cold 0.015 M sodium methoxide in dry methanol. The mixture was kept in a corked tared tube, with occasional shaking, for 3 to 4 h at 0°C. Carbon dioxide then was bubbled through the mixture for 1 to 2 min, and the solvent was evaporated at 25-30°C in a Rotovap apparatus (Buchler Instruments, Fort Lee, N. J.). The residue was treated with benzene and, after evaporation of solvent, was further dried in a vacuum oven (3O”C, 45 min) and stored in a vacuum

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5944 Hydrolytic and Nonhydrolytic Actions of ,B-Amylase

desiccator at -20°C until processed further. The product (average yield, 155 mg, six trials) showed on tic (absolute ethanokethyl acetate, 2:5) a major spot (RF - 0.45) accompanied by a smaller spot (RF - 0.55).

For purification, samples of the deacetylated product (-155 mg) were chromatographed on columns (1.6 X 30 cm) of Silica Gel 60 with 2:5 absolute ethanol:ethyl acetate as developer, as described for a- maltosyl fluoride (16). Fractions (3 ml) were collected and, after testing 50 ~1 of each by the phenolsulfuric acid method (25), those forming the carbohydrate peak were checked for purity by tic. Single- spot (RF = 0.45) fractions were concentrated at 25”C, further dried in a vacuum over (3O”C, 45 min), and stored in a vacuum desiccator at -20°C. Preparations of the final product (average yield, 70 mg from 255 mg of acetate) were chromatographically pure but carried appre- ciable associated solvent as indicated by the carbohydrate content (80 to 88’?& as n-glucose (25)) found for different samples. In the present work, stated concentrations of P-maltosyl fluoride are based on the measured carbohydrate content (25) of the sample used. On this basis, P-maltosyl fluoride was found to have [a]?? + 122” (c 0.26, water).’

Methyl P-M&o&e-Hepta-0-acetyl n-maltosyl bromide (8.2 g) in 200 ml of dry methanol was shaken with 7.5 g of silver carbonate for 12 h in the cold (30). On concentration of the filtrate under vacuum (35°C) to -50 ml, a heavy precipitate of birefringent crystals of hepta-0-acetyl methyl /3-maltoside appeared. This compound (7.6 g; m.p. 128-129°C after recrystallization from absolute ethanol (lit- erature value (31), m.p. 128-129°C)) was deacetylated in 50 ml of 0.01 M sodium methoxide in dry methanol by refluxing for 1 h. The cooled mixture was decolorized with carbon, the solvent was evaporated under vacuum, and the product was dissolved in 20 ml of boiling ethanol. On cooling and seeding with a few crystals of methyl p- maltoside (kindly furnished by Dr. J. Lehrfeld), crystallization began almost immediately and within 10 min at 25°C provided a heavy slurry of long birefringent needles. The dried product (2.0 g) had m.p. 105-109°C and [a]?” + 79.3’ (c 1, water) (methyl /3-maltoside (31): m.p. 110-Ill’C [(Y]:P” + 83.9” (c 2, water).

RESULTS

/?-Maltosyl fluoride, a previously unreported compound, was prepared by way of its crystalline heptaacetate. Unam- biguous assignment of the ,l?-anomeric configuration and D- glucopyranosyl structure of the fluorine-bearing ring of the latter compound was afforded by the “F chemical shift at + 136.4 ppm and coupling constants (J,, F = 53.8 Hz; J2, F = 9.7 Hz) found on ‘“F NMR spectroscopy. These values agree closely with those reported by Hall et al. (32) for tetra-O- acetyl p-n-glucopyranosyl fluoride. ‘H and 13C NMR spectra and elemental analyses likewise agreed with the assigned structure of the compound.

/?-Maltosyl fluoride preparations had the following proper- ties. Treatment with 0.02 N sulfuric acid (lOO°C, 10 min) caused essentially complete hydrolysis of the C-F glycosylic bond. Maltose and glucose were the only products detected on chromatograms: 0.90 to 0.92 mol of fluoride anion was released by the mild acid hydrolysis/2.0 mol of total glucose (25) contained in the sample. The free fluoride content of freshly prepared P-maltosyl fluoride was -1% of the total fluorine present, and this value remained essentially un- changed in samples stored for weeks or months at -20°C in a vacuum desiccator over calcium sulfate. Storage in a desic- cator at ambient temperature was found insufficient to protect against degradation of the compound with release of fluoride. When freshly dissolved in 0.05 M acetate buffer of pH 5.6, p- maltosyl fluoride suffered -0.5% hydrolysis in 30 min at O”C, and -4% hydrolysis in 30 min at 30°C. Because of this lability, all experiments were kept to short duration, and all enzymic reactions were rigorously controlled for the extent of fluoride release in concurrently incubated mixtures with buffer alone.

Hydrolysis of ,&Maltosyl Fluoride by ,&Amylase, and Its Kinetic Distinction from the Hydrolysis of a-Maltosyl Fluo-

’ The corresponding value for a-maltosyl fluoride, [a]z’sV+ 152’ ( c 0.30, water) may be compared with that reported (16) on a weight basis, [a]?’ + 137.4” ( c 0.6, methanol).

ride by the Enzyme-The capacity of crystalline sweet potato /?-amylase to catalyze cleavage of the C-F glycosylic bond of P-maltosyl fluoride, with formation of maltose and hydrogen fluoride (18), was first demonstrated as follows. A mixture containing 0.5 mg/ml of the a-glucosidase-free enzyme, 30 InM

/?-maltosyl fluoride, and 0.05 M acetate buffer of pH 5.6 was incubated at 30°C for 30 min, along with a substrate/buffer control. On chromatography of 5 ~1 of each mixture (50 pg of carbohydrate) uersus glucose and maltose standards, the en- zyme digest showed a very large spot corresponding to maltose (-20 pg as against -2 pg in the control) and also a much smaller P-maltosyl fluoride spot (Rglc 1.17) than the control. Analyses made with a fluoride ion probe further showed the presence of 12.3 ITIM free fluoride in the enzymic digest and 1.3 IIIM in the control. Thus, under the test conditions, /3- amylase catalyzed the breakdown of 11 mM (37%) substrate. Incubation of the enzyme with 5 mM P-maltosyl fluoride under the same conditions led to the production of 0.9 mM free

fluoride above the control level, corresponding to breakdown of 18% of the substrate.

On the basis of these results, study was undertaken of the mechanism whereby ,&amylase acts to produce maltose and hydrogen fluoride from P-maltosyl fluoride, and its relation- ship to the process whereby these products are formed from a-maltosyl fluoride by the enzyme (16, 18). The unusual finding noted above, that a greater percentage of 30 mM than of 5 IIIM P-mahosyl fluoride was utilized upon equal exposure to enzyme, led us to examine the action of P-amylase on /3- maltosyl fluoride (in comparison to that on a-maltosyl fluo- ride) with respect to the relation between initial reaction velocity and substrate concentration. Thus, digests of two series were examined. Those of one series contained 2 to 30 mM a-mahosyl fluoride, 0.01 mg/ml of P-amylase, and 0.08 M acetate buffer, pH 4.85. Those of the second contained 2 to 30 mM P-maltosyl fluoride, 0.25 mg/ml of ,@-amylase, and 0.05 M

acetate buffer, pH 5.6. Individual digests and appropriate substrate/buffer control mixtures were set up at 2-min inter- vals; incubated at 30°C for 36 min, then analyzed for fluoride ion content (in all cases, substrate utilization was less than 21%). Initial specific rates of enzymically catalyzed fluoride release were’calculated after correction for a- or /?-maltosyl fluoride breakdown (0.34 and 5.4%, respectively) in the con- trols incubated without enzyme.

It is clear from the results (Fig. 1) that a great difference exists between the actions of P-amylase on a- and ,&maltosyl fluoride. In the case of the a anomer, a plot of the initial velocity of fluoride release, u, as a function of substrate con- centration, S, has the hyperbolic form usually associated with direct hydrolytic reactions; also, a linear Lineweaver-Burk relationship is obtained (16). In contrast is the upward concave curvature of a plot of the rate of fluoride ion release from ,& maltosyl fluoride uersus substrate concentration, likewise that of the plot of v-l versus S-i. Fig. la shows that between 2 and 7.5 mM substrate the rate of fluoride release from a- maltosyl fluoride rose steeply, but in decelerating fashion, to a value of 1.28 pmol/min/mg, whereas (Fig. lc) the rate from ,Kmaltosyl fluoride rose slowly, in accelerating fashion, to only 0.10 pmol/min/mg at the 7.5 mM substrate level. Between 12 and 30 mM, the speed of fluoride release from a-maltosyl fluoride remained essentially unchanged at -1.5 pmol/min/ mg; in contrast, P-maltosyl fluoride was utilized with ever increasing speed in this range, e.g. at rates of 0.27 and 0.69 pmol/min/mg at 15 and 30 mM.Z That this behavior of the ,& maltosyl fluoride-/3-amylase system represents activation by

’ P-Amylase was found to utilize 45 mM /?-maltosyl fluoride at a rate of 2.7 pmol/min/mg (experiment of Fig. 3), and 53 mM P-maltosyl fluoride at an initial rate of 3.8 pmol/min/mg (experiment of Fig. 4).

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Hydrolytic and Nonhydrolytic Actions of /3-Amylase 5945

a second molecule of substrate was further indicated by the observation that an essentially linear relationship between u-l and S2 obtains at concentrations of /3-maltosyl fluoride below 6 mM. These results might possibly be due to a homotropic allosterism (33); however, a further possibility is that the action of P-amylase on fi-maltosyl fluoride does not directly involve water as a reactant but may require 2 molecules of substrate, one to function as a maltosyl donor and the other as a maltosyl acceptor.

Enhanced Enzymic Utilization of /3-Maltosyl Fluoride in the Presence of Structurally Related Carbohydrates-The idea that P-maltosyl fluoride might be utilized by a process other than direct hydrolysis was supported by a further find- ing: that certain compounds having a terminal D-ghCOpyG+

“:;zi j/F3

0 IO 20 30 S

-.2 ys .2 .4

.6

.4 V

El

. /

.2 /

2 C

-.2 , .2 .4

FIG. 1. Difference in the kinetics of hydrolysis of (Y- and P-maltosyl fluoride by crystalline sweet potato /3-amylase. a, a-Maltosyl fluoride: rates of fluoride anion release (in micromoles/min/mg of protein) at 30°C and pH 4.8 versus substrate concentration (in micromoles/ml). 6, a-Maltosyl fluoride: relationship between l/u (in micromoles of F- released/min/mg of protein)-’ and l/S (in micromoles of substrate/ ml)‘. c, /?-Maltosyl fluoride: rates of fluoride anion release (in micro- moles/min/mg of protein) at 30°C and pH 5.6 uersus substrate concentration (in micromoles/ml). CE, /3-Maltosyl fluoride: relationship between l/u (in micromoles of F- released/min/mg of protein)-’ and l/S (in micromoles of substrate/ml))‘.

0.4

3 + 5 0.3

d

F 1 5 0.2 8’ lL

4 s” a 0.1

0

A

t50 mM METHYL-B-MALTOSIDE

nosy1 moiety enhance the ,f3-amylase-catalyzed release of flu- oride from P-maltosyl fluoride. Fig. 2A shows the effect of one such compound, methyl ,&maltoside, on the rate of fluoride ion liberation at several low substrate concentrations. Findings are presented for two concurrently prepared and analyzed series of ,&maltosyl fluoride-b-amylase digests, one without and one containing methyl j?-maltoside. The open circle curve has the upward concave form regularly found with P-maltosyl fluoride as the sole substrate. The closed circle curve ap- proaches linearity and shows the enhanced rates of fluoride release (g-fold at the lowest substrate concentration tested) found in the presence of 50 mM methyl P-maltoside. Incuba- tion with this glycoside did not cause any liberation of fluoride from ,f?-maltosyl fluoride in the absence of /I-amylase. Fig. 2 B illustrates the results of a similar experiment in which 25 mM

p-nitrophenyl cY-n-glucopyranoside was added as the “substi- tute acceptor.” Enhancement of fluoride ion release, although not as pronounced as with 50 mu methyl P-maltoside, is clearly present (a &fold increase in rate was produced at the lowest substrate concentration). In other experiments, 25 mM

maltose was also found to augment, although to only a small degree, the release of fluoride from P-maltosyl fluoride by /?- amylase. No enhancement was detected with phenyl or p- nitrophenyl P-n-glucoside, sorbitol, mannitol, or glucose.

Polarimetric Determination of Configuration of the Mal- tose Formed from a- and ,&Maltosyl Fluoride by ,%-Amylase Action-To more fully characterize the reactions catalyzed by P-amylase from (Y- and /3-maltosyl fluoride, a polarimetric study was made to ascertain the anomeric configuration of the maltose which is produced from each. In one case, optically clear solutions of 10.2 m&r a-mahosyl fluoride in 0.08 M acetate buffer of pH 4.8, and of 1.4 mg/ml of P-amylase (Sigma) in the same buffer, were brought to 26°C and admixed in equal volume. Part of the digest was introduced into the lo-cm cuvette of a Perkin-Elmer model 141 photoelectric polarime- ter and the rotation, (uz’” followed during the fist 18 min of reaction. The remainder of the digest, also held at 26”C, was used to determine the concentration of fluoride released at intervals during this reaction period. Similarly, optically clear solutions of 90 mM P-maltosyl fluoride and of 4.2 mg/ml of j?-amylase in 0.1 M acetate buffer of pH 5.6 were attempered at 25°C and mixed in equal proportions. The digest was analyzed for a?y, using a I-cm cuvette, and for fluoride release at intervals during the fust 7.5 min of reaction.

El

+ 7.5 mM PNPo(-D-GLLICOSIDE

FIG. 2. Enhanced utilization of p- maltosyl fluoride by /I-amylase in the presence of glycosides that presumably act as substitute maltosyl acceptors. Spe- cific rates of fluoride anion release for paired series of miztures containing 0.85 to 6.8 mM /?-maltosyl fluoride, 0.15 mg/ mf of ,L?-amylase, and 0.05 M acetate buffer, pH 5.6. Mixtures with and with- out added glycoside were set up alter- nately, at 2-min intervals, and analyzed for fluoride content after incubation at 30°C for 24 min; substrate/buffer and substrate/buffer/glycoside controls were included. A, rates in the presence (0) or absence (0) of 50 mM methyl /3-malto- side B, rates in the presence (W) or ab- sence (0) of 25 mM p-nitrophenyl a-~- glucopyranoside.

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p- MALTOSYL FLUORIDE, mM

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5946 Hydrolytic and Nonhydrolytic Actions of ,8-Amylase

Fig. 3 shows the observed optical rotation with time for each digest, together with the rotations expected in the event a-maltose (or p-maltose) was the reaction product present. The latter values were calculated on the basis of the molar concentration of fluoride (hence, of maltose) released during enzymic digestion. The results in Fig. 3 (upper part) show clearly that the maltose produced by the action of /?-amylase on a-maltosyl fluoride (16) is the p anomer. Those in Fig. 3 (lower part) indicate that the maltose released in the digest of ,&maltosyl fluoride also is the ,B anomer. If, as appears to be the case, p-amylase catalyzes the formation of identical products (/?-maltose and hydrogen fluoride) from both a- and ,f?-maltosyl fluoride, the reactions of hydrolysis of the two anomers would have to occur by different paths. As this point is a critical one, and as configurational inversion is considered a hallmark of reactions catalyzed by ,L?-amylase, confirmation of the form of maltose produced from ,&maltosyl fluoride was sought using an independent and more specific method.

‘H NMR Determination of Configuration of the Maltose Produced from ,&Maltosyl Fluoride by /3-Amylase-The like- lihood that 100 MHz Fourier transform ‘H magnetic resonance spectroscopy might yield definitive information on the ano- merit form of the maltose produced by @-amylase acting on /?-maltosyl fluoride was indicated by the results obtained with reference spectra, which showed that the anomeric protons of substrate and of either potential product have readily distin- guishable resonances. Thus, 20 I’UM ,&maltose, before and after anomerization, gave chemical shifts and coupling con- stants of 4.64 ppm and J,, 2 = 8.0 Hz for the C-l axial proton of the reducing n-glucose residue; 5.24 ppm and J1,2 = 3 Hz

o( -MALTOSYL FLUORIDE, 5.1 mM ,3,.F 2p y3XHYcROLYS/

48 IOcm PATH

48 I cm

PATH

0 4 0 12 16

DIGESTION (26.1, MINUTES

p-MALTOSYL FLUORIDE, 45 mM 25 50 X HYDROLYSIS

----a-- ----A

,&- O(-MALToSE

~-MALTOSE

0 2 4 6 6

DIGESTION (25.1, MWES

FIG. 3. Apparent production of the /I-anomer of maltose from both (Y- and P-maltosyl fluoride by the action of crystalline sweet potato P-amylase. A, observed optical rotations (0) of a digest costaining 5.1 mM a-maltosyl fluoride and 0.7 mg/mI of P-amylase in 0.08 M acetate buffer, pH 4.8; observed rotation (0) of 5.1 mM cu-maltosyl fluoride in the same buffer, without enzyme. B, observed optical rotations (W) of a digest containing 45 mu /I-maltosyl fluoride and 2.1 mg/ml of p- amylase in 0.1 M acetate buffer, pH 5.6; observed rotation (0) of 45 mu b-maltosyl fluoride in the same buffer, without enzyme. In each case, calculated optical rotations (A) are based on the concentration of fluoride anion released, assuming the product to be entirely CX- maltose, [alo + 174’, or entirely p-maltose, [(U]~ + 120”.

lax

u

X

L

I I I I 6 5 4 3 ppm

FIG. 4. ‘H nuclear magnetic resonance spectra (100 MHz) of a p- maltosyl fluoride-P-amylase digest (22”C, pD 5.7) at 1 to 5 min (A), 16 to 20 min (B), and 3 h (C). 1ax, C-l axial proton of the reducing n-glucose moiety of p-maltose (4.64 ppm); leq, C-l equatorial proton of the reducing n-glucose moiety of a-maltose (5.24’ppm); S, C-l equatorial proton of the fluoride-bearing D-ghCOSe moiety of the substrate, P-maltosyl fluoride (5.25 ppm); nr, C-l equatorial proton of the nonreducing n-glucosyl moiety of P-mahosyl fluoride, maltose, or both (5.40 ppm); x, spinning side band artifact. Resonances are measured downfield from 3-(trimethylsilyl)propanesulfonic acid so- dium salt.

for the C-l equatorial proton of the reducing D-ghCOSe residue; 5.40 ppm and JI, 2 < 3 Hz for the C-l equatorial proton of the nonreducing D-glucose moiety. These values are in agreement with those reported by Rao and Foster (34). Spectra obtained with 40 mM P-maltosyl fluoride gave values of 5.26 ppm, JH,, “2 = 7.3 Hz, and JH~.F = 52 Hz for the C-l axial proton of the fluorine-bearing D-glucose moiety, in agreement with the val- ues reported by Hall et al. (32) for tetra-0-acetyl ,&D-gluco- pyranosyl fluoride, and also values of 5.44 ppm and Jlz < 3 Hz for the C-l equatorial proton of the nonreducing D-&COSe

moiety. For examination of the P-amylase catalyzed hydrolysis of

fi-maltosyl fluoride, both enzyme and substrate were treated to exchange their labile hydrogens for deuterium atoms. The P-amylase (1140 unita/mg, Sigma) was exhaustively dialyzed at 8°C versus 0.2 M acetate-dJdeuterium oxide buffer of pD 5.7. The /3-maltosyl fluoride was dissolved in methanol-d4 and recovered in dry form just before use by removing the solvent in a vacuum evaporator at 25°C. At time zero, a digest was prepared by adding 1.2 ml of the dialyzed p-amylase (2.1 mg/ ml in 0.2 M acetate-d,, buffer of pD 5.7) to 63.6 pmol of the deuterium-exchanged ,&maltosyl fluoride. Part of the mixture was transferred to a 5-mm NMR tube, and ‘H NMR spectra were recorded at intervals during 3 h at 22°C; each spectrum consisted of 128 free induction decays using 2-s repetition times. The remainder of the digest, also kept at 22’C, was

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Hydrolytic and Nonhydrolytic Actions of /3-Amylase 5947

used to determine the concentration of fluoride liberated at intervals up to 20 min.

Fig. 4 shows the NMR spectra. Resonances at 4.64 ppm (J,,l = 8 Hz), attributable to the C-l axial proton of the reducing n-glucose residue of p-maltose, are present at 5 min (Spectrum A), and are maximal at 20 min (Spectrum B) when the enzymic reaction was nearly complete (49.4 mM hydrogen fluoride released from 53 mM P-mahosyl fluoride). Resonances of the C-l equatorial proton of the reducing D-ghCOSe residue of a-maltose (at 5.24 ppm, J1, 2 = 3 Hz) are not seen at 5 min (Spectrum A) and are just detectable at 20 min (Spectrum B). By 3 h, anomerization has occurred, and strong signals for both equatorial and axial protons of the reducing D-&COW

unit are present (Spectrum C). Resonances at 5.25 ppm, referable to the anomeric proton of the fluorine-bearing D-

glucosyl residue of the substrate, are initially strong (Spec- trum A) and are greatly diminished by 20 min (Spectrum B).

DISCUSSION

The present study provides a unique demonstration of the ability of a carbohydrase to utilize both anomeric forms of a substrate. The observed hydrolytic conversions of a- and /?- maltosyl fluoride by purified sweet potato P-amylase, with production of p-maltose and hydrogen fluoride in each case, show this enzyme to have a catalytic capability not previously recognized for any carbohydrase. As discussed below, eluci- dation of the paths of these reactions reveals that P-amylase is the catalyst of two complementary types of reactions rather than of hydrolysis alone; in turn, it would appear that the enzyme’s functional groups in the active site have dual (rather than single) roles which would be reversed in promoting the two types of reactions. We have recently reported similar findings of widened catalytic scope and dual functionality of the catalytic groups of a- and ,8-glucosidase (12) in a nearly opposite situation in which these two enzymes were found to bring about stereospecific hydration of the same nonglycosidic substrate, D-ghCd. In short, a new level of understanding of ,&amylase action has been obtained through the study of glycosylation reactions catalyzed without glycosidic bond cleavage, as also has recently been found for a- and ,&gluco- sidases (12), /?-galactosidase (13, 14), and a-amylases (15, 17). The ability of these extensively characterized, classic carbo- hydrases to catalyze glycosylation reactions with glycosyl fluoride and enolic glycosyl substrates confiis the thesis advanced by Hehre et al. (11) that glycoside hydrolases and glycosyl transferases are glycosylases, all of whose varied reactions effect a uniform and well defined chemical event, the interchange of a glycosyl residue and a proton.

Insight into the different pathways of hydrolysis of a- and P-maltosyl fluoride by /3-amylase was provided by the kinetics of cleavage by the enzyme of the C-F glycosylic bond of these substrates. Examination of the rate of fluoride release as a function of substrate concentration shows the complexity of the enzymic hydrolysis of ,&maltosyl fluoride compared to that of a-maltosyl fluoride. With the latter substrate, the relation of reaction velocity to substrate concentration was found to be completely in accord with direct hydrolysis of the substrate as recently reported (16). In contrast, the reaction catalyzed by ,f3-amylase with ,&maltosyl fluoride was charac- terized by upward concave curves of the relationship between v and S, and between v-’ and S’, indicative of activation by substrate. In addition, a linear relationship was observed between v-’ and S” at low concentrations of /?-maltosyl fluoride, further suggesting that the activation may be com- pulsory and may involve binding of 2 molecules of substrate (33, 35). For reasons discussed below, it appears unlikely that these kinetic results represent allosteric effects. Rather, we

envision that the enzymic reaction may require the presence of 2 molecules of /3-maltosyl fluoride at the catalytic site, which serve as maltosyl donor and acceptor. This interpreta- tion accounts for the further unique finding that certain compounds resembling the substrate in having a terminal a- D-glucopyranosyl moiety (e.g. methyl fl-maltoside and p-ni- trophenyl-a-n-glucopyranoside) greatly increase the rate of utilization of ,&maltosyl fluoride by P-amylase. These com- pounds would appear to serve as substitute acceptors or “primers” for ,8-amylase-catalyzed transfer reactions when p- maltosyl fluoride is present in low concentration. Other ex- perimental results also fit best with the view that an acceptor other than water is required for /?-amylase to act upon p- maltosyl fluoride, even though the result finally observed is a hydrolytic conversion.

The reaction pathways that can account for the actions of P-amylase on a- and ,&maltosyl fluoride, to yield p-maltose and hydrogen fluoride, are given in Scheme 1. For comparison, paths are shown (Equation A) for (I) the hydrolysis of the central a-1,4-n-glycosidic linkage of maltotetraose to give p- maltose and (II) for the reversal of this reaction. As demon- strated by Hehre et al. (19), reversal involves enzyme-cata- lyzed maltosyl transfer from the ,8 anomer of maltose to the C-4 hydroxyl of a second molecule of maltose, to produce maltotetraose. Equation B illustrates the hydrolysis of the C-F glycosylic bond of a-maltosyl fluoride, with the forma- tion of ,&maltose. This reaction is envisioned as proceeding along the same path (I) as the hydrolysis of maltotetraose on the basis of the a anomeric configuration of the substrate, normal kinetic course of the reaction, and inverted (p-) con- figuration of the product. Equations C and D show the path- way for the ,&amylase-catalyzed hydrolysis of ,&maltosyl flu- oride to p-maltose, indicated as occurring by way of two discrete steps. The first step, C, involves cleavage of the C-F glycosylic bond of /?-maltosyl fluoride, with transfer of the maltosyl moiety to a second substrate molecule to form mal- totetraosyl fluoride. This step is viewed as occurring along the same reaction path (II) as the enzymic condensation of /3- maltose to maltotetraose (cfi A). This is in agreement with the kinetic findings which suggest the need for 2 molecules of substrate to be bound by the enzyme for C-F bond cleavage to occur. The presumed a-maltosyl intermediate produced by this step then is rapidly hydrolyzed to give p-maltose through reaction D which follows the same path (I) as the hydrolysis of maltotetraose. The combination of these two sequential steps thus accounts both for the kinetic findings (including enhanced enzymic utilization of ,&maltosyl fluoride in the presence of substrate-related compounds), and for the ob- served retention of configuration in the overall hydrolytic conversion.

It should be mentioned that /?-maltotetraosyl fluoride has thus far not been detected in the reaction of P-maltosyl fluoride with beta amylase. Only minute amounts of this intermediate, however, would be expected in steady state during the reaction since only an extremely small proportion of maltotetraose is found in equilibrium with ,&maltose in the presence of the enzyme (19). One might wonder if ,&maltosyl fluoride might be directly hydrolyzed to p-maltose without mediation by an a-maltosyl transfer product, assuming that the activating effect of substrate is exerted from a site on the enzyme that is removed from the catalytically active groups. This path, however, seems most unlikely as it fails to account for the observed synthesis of maltotetraose from ,&maltose catalyzed by the enzyme. Thus, although the conversion of ,&maltosyl fluoride to p-maltose is exceptional in that no reaction catalyzed by P-amylase has hitherto been found to produce retention of configuration (2-10, 19, 36), this demon-

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5948 Hydrolytic and Nonhydrolytic Actions of P-Amylase

I A cY-Maltosyl-1,4-maltose + HZ0 ‘II p-maltose + maltose

B a-Maltosyl-F + H20 I

-+ p-maltose + HF

C P-Maltosyl-F + /3-maltosyl-F % a-maltosyl-1,4-b-maltosyl-F + HF

D -Hz0 1 +HzO

III I I

p-maltose + P-maltosyl-F

SCHEME 1

stration of configurational retention by the enzyme can be reconciled with past experience. All available evidence indi- cates that the conversion proceeds as in Scheme 1, by two successive reactions, each producing inversion.

The sequential formation and hydrolysis of glycosyl transfer products in the same digest is well known for glycosidases and glycanases that catalyze reactions with retention of configu- ration (15, 17, 37-40). However, it has been widely reported that “inverting exo-glycan hydrolases,” such as ,8-amylase (4, 6, 9, 20), glucoamylase (20, 41-43), exo-P-1.3-glucanase (44), papaya lysozyme (45), glucodextranase (46, 47), etc., do not catalyze glycosyl transfer reactions. Past failures to demon- strate the ability of these enzymes to catalyze glycosylation reactions in which water is not a co-substrate or product have invariably involved the use of donor substrates with glycosyl residues of anomeric configuration suitable only for the hy- drolytic reaction. Based on the present findings with P-amy- lase and P-maltosyl fluoride, the observation of such reactions would appear to require the use of appropriate substrates of the opposite configuration. Glycosyl transfer reactions (i.e. glycosylations in which water is not a participant), accompa- nied by inversion of configuration, are catalyzed by various disaccharide and nucleoside phosphorylases, P-glycan syn- thases, etc. The present instance shows that this capacity is not restricted to such enzymes but extends to the prototype of the inverting exoglycanases.

It is apparent, as indicated in Scheme 1, that /3-amylase has the capacity to catalyze two different types of glycosylation reactions: namely, hydrolysis of a-1,4-n-glycosidic substrates’ and a-maltosyl fluoride to form p-maltose, and maltosyl trans- fer from p-maltose and P-maltosyl fluoride to carbohydrate acceptors to form a-1,4-n-glycosidic products. The stereospe- cihcahy complementary nature of these two types of reactions provides insight into the enzyme’s catalytic mechanism, heretofore reported only in terms of the hydrolysis of a-1,4- D-glycosidic linkages. Evidence exists for the presence of an imidazole and a carboxyl residue in the active site of /?-amylase (48, 49). As discussed by Thoma and co-workers (10, 49, 50), the imidazole group is presumed to function as a source of protonation of the glycosidic oxygen atom of cu-1,4-linked substrates, with the carboxyl group existing in the ionized state and functioning either as a general base, an electrostatic stabilizing group, or a nucleophile, Scheme 2A (I) illustrates such a mechanism for the hydrolysis of maltotetraose in which the imidazole group acts as a general acid and the carboxylate anion is assumed (for reasons given below) to act as general base assisting the attack of a water molecule on the substrate. In Scheme 2B the same mechanism is illustrated with respect to the hydrolysis of cr-maltosyl fluoride. Scheme 2C shows the postulated mechanism of the reaction of @-amylase with /3- maltosyl fluoride. The apparent requirement for the presence of 2 molecules of P-maltosyl fluoride at the active site in order for C-F bond cleavage to occur strongly suggests that the

fluorine atom of the donor substrate molecule is displaced by the C-4 hydroxyl of the second substrate molecule located at the acceptor site. This favors a concerted mechanism in which imidazole acts as a general base or specific base to assist the C-4 hydroxyl group of the acceptor to displace the /?-linked fluoride, in turn assisted by general acid catalysis by the carboxyl group. This mechanism is distinguished from one in which the fluoride ion of the donor substrate departs fist, leaving a carbonium ion that is subsequently captured by the C-4 hydroxyl of the acceptor. The concerted displacement mechanism for the action of ,8-amylase on /3-maltosyl fluoride suggests, although it does not prove, that the condensation of ,&amylase to form maltotetraose (19) also involves a concerted displacement (Scheme 2A, II). In that case, the principle of microscopic reversibility would require that the hydrolysis of maltotetraose to p-maltose also be concerted, as indicated in Scheme 2A. This would argue against the formation of a free carbonium ion intermediate for the hydrolysis of amylaceous substrates by fi-amylase, a mechanism which has been gen- erally accepted (10, 49-51). However, it is possible that, al- though the displacement reaction of ,&maltosyl fluoride may be a concerted reaction, ,&maltose may undergo a stepwise condensation reaction with the carbonium ion intermediate. These possibilities are not distinguishable at present.

Regardless of the details of the proposed mechanism, it appears that the functional groups in the active site of beta amylase, presumably imidazole and a carboxyl group, must have a wider catalytic capacity than previously assumed. Until now, the functioning of these catalytic groups has been con- sidered essentially in terms of promoting hydrolytic reactions. The catalysis not only of the reverse reaction with p-maltose (19), but also of analogous glycosylation reactions with ,8- maltosyl fluoride which are not reversals of hydrolysis, re- quires catalytic flexibility on the part of these functional groups. Indeed, this important point also emerges from a study of the hydration of n-glucal catalyzed by (Y- and p- glucosidase (12), in that the direction of protonation of this nonglycosidic substrate by each of these enzymes was found to be opposite from that proposed in the currently accepted reaction mechanisms for the hydrolysis of the usual (glycosid- ically linked) substrates. j?-Galactosidase, in catalyzing 2- deoxy-n-galactosyl transfer from D-gdaCta1 to glycerol, like- wise has been shown to protonate this enolic glycosyl sub- strate from a different direction than when acting on B-D-

galactosides (14). Furthermore, Rupley et al. (52, 53) have provided evidence that glutamic acid 35 in hen egg white lysozyme functions as a general base catalyst in transglyco- sylation reactions, and as a general acid in hydrolytic reac- tions.

In light of the above observations, we propose a general mechanistic model for the action of glycosidases that empha- sizes the catalytic flexibility of their active site functional groups in effecting reactions according to the pattern, glycosyl-

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Hydrolytic and Nonhydrolytic Actions of ,&Amylase 5949

0

SCHEME 2

X+ H-X’= glycosyl-X’ + H-X (l), where -X and -X’ are sites of glycosyl-proton interchange. This model encompasses more than consideration of the reversed catalytic functions of the active site groups of these enzymes based on the principle of microscopic reversibility. Rather, it accounts not only for the ability of glycosidases to effect hydrolytic and corresponding reverse (condensation) reactions but also for their ability to catalyze the further types of glycosylation reactions discussed above. The proposed model, finally, provides a mechanistic basis in support of the view that glycosidases and glycosyl transferases are interrelated members of an inclusive class of enzymes designated as glycosylases ( 11).

Acknowledgments-We express our thanks to Drs. Otto Wurzburg and Leo Kruger of the National Starch and Chemical Company for their kindness in making available a Perkin-Elmer 141 photoelectric polarimeter, and to Dr. Kruger for his kind help with the polarimetric measurements recorded in Fig. 3. Thanks are due, also, to Drs. J. J. Marshah and W. J. Whelan for the generous gift of a-glucosidase-free /3-amylase and to Dr. J. Lehrfeld for a sample of pure methyl /3- mahoside.

9. 10.

11.

12.

13.

14. 15.

16.

17.

18.

19.

1.

2.

3. 4.

5. 6. 7.

8.

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E J Hehre, C F Brewer and D S Genghofactions of beta-amylase on alpha- and beta-maltosyl fluoride.

Scope and mechanism of carbohydrase action. Hydrolytic and nonhydrolytic

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