TJIE JOURXAI. OF BIOLOGICAL CHEMISTRY

Vol. 21i, So. 2, Issue of Junuilry 25, pp. 532-512, 1972 Printed in U.S.A.

Mechanism of Action of Sucrose Phosphorylase

III. THE IZEACTION WITH WATER An;D OTHMt ALCOHOLS*

(Received for publication, July 1, 19 1)

JOHS J. MIEYAL,$ MARCIA SIMOK, AND ROBERT H. ABELES

FI-011% the Graduate Department of Biochemistry, Branrleis University, W’alfham, Massachusetts 0~?154

SUMMARY

Sucrose phosphorylase is active in various alcohol-water mixtures and can catalyze the transfer of the glucosyl moiety of sucrose to the alcohols forming the corresponding glu- cosides. The efficiency of the glucosyl acceptor (V,:n;Lx/Kn) decreases in the order: rrons-1,2-cyclohexanediol (0.057) M ethylene glycol (0.061) > methanol (0.017) NN cis-1,2- cyclohexanediol (0.015) > ethanol (0.002) > water (0.001). The dependence of the rates on the concentration of each of the alcohols yields kinetics which shows saturation suggesting that each of the alcohols binds to the enzyme. The apparent V,,, for most of the alcohols is similar to the hydrolytic rate. frans-1,2-Cyclohexanediol and ethylene glycol react with the glucosyl enzyme without reducing the rate of hydrolysis although the water concentration is significantly reduced as the concentration of the alcohol is varied (e.g. 0 to 30% ethylene glycol by volume). frans-1,2-Cyclohexanediol is competitive with phosphate and noncompetitive with sucrose. Ethylene glycol is noncompetitive with both phosphate and sucrose. cis-1,2Xyclohexanediol and methanol reduce the rate of hydrolysis. The reaction with methanol yields ac-methylglucoside, and its rate of formation is apparently not reduced by phosphate. It was concluded that two separate, independent binding sites exist. These were designated water site, at which water and probably methanol react, and acceptor site, at which phosphate and the sugar acceptors as well as frans-1,2-cyclohexanediol and ethylene glycol react. The site of interaction of cis-1,2-cyclohexanediol is un- certain. Two adjacent hydroxyl groups on the glucosyl acceptor molecule seems to be a necessary, but possibly not a sufficient property for interaction at the acceptor site.

Sucrose phosphorylase (disaccharide glucosyltransferase, EC 2.4.1. a), isolated from Pseudomonas saccharophila which had been induced with sucrose, primarily catalyzes the transfer of

* This work was supported by Grant GB-16413 from the Na- tional Science Foundation and a United States Public Health Service Postdoctoral Fellowship No. 1 F02 GM-42,919.01 (J.M.). Publication No. 809 from the Graduate Department of Bio- chemistry, Brandeis University, Waltham, Mass. 02154.

1 Present address, Departments of Pharmacology and Bio- chemistry, Northwestern University Medical School, Chicago, Ill. 60611.

the glucosyl moiety- of sucrose to inorganic phosphate forming fructose and a-glucose-l-P. There is no net inversion of con- figuration at the C-l atom of the glucoside (l), but IsO studies (2) have shown that the glycosidic bond is cleaved between the

C-l atom of the glucosyl moiety and the glycosidic oxygen atom. The reaction is reversible; i.e. glucose-l-l” can act as glucosyl donor and fructose as acceptor, and the enzyme catalyzes the exchange of [14C]fructose into sucrose (3) and of 32Pi into glucose- 1-P (4). These findings, along with kinetic data (5), indicative of a ping-pong mechanism, and the isolation of a covalent glucose-enzyme in which the glucosyl moiety is bound to the enzyme by a p linkage (6) have formed the basis for the conclu- sion that the over-all reaction takes place via a double replace- ment mechanism involving a glucosyl enzyme intermediate.2 Consistent with this conclusion is the fact that the enzyme is highly specific for the a-D-ghICOSyl moiety; on the other hand, D-xylulose, L-sorbose, u-rhamnulose, L-arabinulose, and L-arabi- nose can each replace fructose as glucosyl acceptor and form the corresponding disaccharide reversibly. According to the inter- pretation of Gottschalk (8), the only apparent requirement for the glucosyl acceptor (Ivhether it be a five- or six-membered ring) is that the hydrosyl group on the carbon atom (C-3) adja- cent to the glycosidic bond be &disposed to the oxygen atom of the glycosidic bond. brsenolysis of both sucrose and glucose- 1-P also occurs, but yields glucose and fructose (or Pi) irrevers- ibly (4, 5, 9). Det)ails of the above observations and conclu- sions have been reviewed (10-12).

Besides the transfer reactions listed above, sucrose phospho- rylase also catalyzes the hydrolysis of sucrose, glucose-l-P and glucose l-fluoride (5, 9, 13), but the hydrolytic reaction proceeds nearly two orders of magnitude slower than the phosphorolytic reaction and with a pH optimum nearly 1 pH unit lower (5). Furthermore, although the rates of phosphorolysis of sucrose, glucose-l-P and glucose l-fluoride differ by as much as 3-fold (5, 13), the rates of hydrolysis of these same glucosyl donors are approximately equal (5, 13). Nevertheless, it has been shown that the hydrolytic reaction is an intrinsic property of the purified enzyme (5, 9). These differences in rates and pH dependence, as well as buffer effects to be described in this communication, suggest that the hydrolytic reaction and the transfer reaction

1 The abbreviations used are: glucose-l-P, a-glucose l-phos- phate; MES, potassium morpholinoethanc sulfonate; glucose&P, a-glucose B-phosphate.

2 Additional evidence implicating a carboxyl group as the glu- cose-binding residue on the enzyme was presented at the Fifty- fourth Annual Meeting of the American Society of Biological Chemists, Atlantic City, April 1970 (7).

532

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Issue of January 25, 1972 J. J. Mieyal, M. Ximon, and R. H. Abeles 533

may hnl-e different rate-determining steps. The question also arises whether the reactions with water and with other acceptors occur at the same site. If not, is there a separate water-bind- ing site or does water react directly from solution? These questions can be answered, at least in part, by exploring the effect of acceptors, such as Pi, on the hydrolytic reaction. This approach is experimentally difficult because of the large dif- ference in rate between the hydrolytic and phosphorolytic re- actions. To skirt this difficulty we examined a number of alco- hols for acceptor capability. It was found that several alcohols can function as glucosyl acceptors and react at rates comparable to the hydrolytic rate. Hence, these alcohols were suitable for investigating the questions posed above. These experiments are reported in the present communication.

EXPERIMEKTAL PROCEDURE

Xaferinls-Sucrose phosphorylase was purified from P. sac- charophiln3 :LS described previously (5). All other enzymes named below for use in assay systems and NADP were obtained from IIorhrillger-Mallrlheim Corp., except for the Glucostat reagent which was obtained from Wort,hington Biochemical Corp. cr-h/Iethylglucoside was purchased from Sigma Chemical Co., n11d P-methylglucoside from Calbiochem. Uniformly la- beled [Y:]sucrosc was obtained from New England Nuclear Corp. Cis- and trnns-1,2-cyclohexanediol were purchased from Frinton Laboratories, Vineland, N. J. All other routinely used chemicals were obtained from various commercial sources and were not, further purified.

~llethods~[‘4C]Sucrose (uniformly labeled) was diluted as fol- lows: 0.25 mCi of [%]sucrose (uniformly labeled) in ethanol solution of specific activity 346 mCi per mmole was transferred t.o a 50.ml ground glass, round bottom flask along with 1.38 ml of a 0.04 nf aqueous solution of unlabeled sucrose. The resulting solution was evaporated to dryness by the technique of bulb-to- bulb vacuum distillation. The dry residue was redissolved by adding 1.38 ml of water. The radioactivity of the final solution was det,ermined by liquid scintillation counting as previously described (5); the sucrose concentration was determined by the phenolsulfuric acid procedure (14).

Sucrose phosphorylase was routinely assayed for enzymatic activity, as described previously (5), by a coupled continuous assay &em monitoring NADPH production.

Rates of formation of individual products were usually detcr- mined by assaying aliquots for that particular product from samples quenched periodically during the course of the reaction. The met,hod of sampling in this case involved distributing the reaction mixture, after addition of sucrose phosphorylase at zero time, into several test tubes. These portions were then boiled at given time intervals by placing the test tube directly into the flame of a Bunsen burner. A similar method of quench- ing was previously used (5) in a kinetic study of this enzyme. Glucose-l-P was assayed spectrophotometrically (5). Glucose IT-as either determined with the Glucostat reagent (Worthington) or spectrophotometrically with hexokinase and glucose-6-P de- hydrogpnase (5). Glucose plus fructose was determined spec- trophotometrically with hexokinase, glucose-6-P dehydrogenase, and phosphoglucose isomerase (5).

The rates of glucose and of glucose plus fructose production were also determined by a continuous assay. For each rate

3 We are grateful to Dr. Thomas H. Stout of the Merck, Sharpe and Dohme Co.. for a generous gift of bacterial cells.

determination, 0.4 ml of assay mix was combined with 0.1 ml of NADP solution (4 mg per ml), 0.25 ml of water or alcohol, 1 /*l of hexokinase (2 mg per ml), 1 ~1 of glucose-B-l’ dehydrogenase (5 mg per ml), and when fructose was also determined 2 ~1 of phosphoglucose isomerase (2 mg per ml). The assay mix con- tained 5 ml of 1 M potassium morpholinoethane sulfonate buffer, pH 6.5, 0.16 g of K2-ATP, 0.5 ml of 0.1 M MgCl*, 0.1 ml of mer- captoethanol, and 14 ml of HZO. An appropriate amount of sucrose phosphorylase was added. The amount of glucoside present was usually determined as the difference between the amounts of fructose and glucose present (i.e. [(glucose + fruc- tose) - glucose] - glucose).

[14C]Methylglucoside was separated from [14C]sucrose, [‘“Cl- glucose, and [14C]fructose by means of descending paper chroma- tography (15) for 15 hours (the solvent pyridine-ethyl acetate- water (5:12:4 v/v/v) was allowed to drip off the serrated edge of the chromatogram). The radioactive compounds were lo- cated on paper by means of a Tracerlab windowless 4?r scanner. Methylglucoside was also separated from the other sugars by means of column chromatography (Dowex l-OH-, 0.5 x 17 cm (16)). The methylglucoside was eluted with water and de- tected by the phenol sulfuric acid, nonspecific carbohydrate assa,y (14). The isolated methylglucoside and authentic samples of oc-methylglucoside and fl-methylglucoside were converted to the corresponding trimethylsilyl derivatives by the method de- scribed previously (6). Trimethylsilyl 01. and P-methylgluco- side were separated by gas chromatography on a column (0.3 x 165 cm) of Chromosorb W (AW-DMC-S) (100 to 120 mesh) containing 3% SE 30. An F & M Scientific Corp. model 720 gas chromntograph, equipped with a thermal conductivity de- tector, was employed at a constant oven temperature of 140”. The retention times of the two anomers under these conditions are separated by 3 min. The F & M gas chromatograph is coupled by means of a stream splitter to a Nuclear Chicago gas flow radioactivity counter. In this way, it can be determined to which peak on the gas chromatogram radioactivity is associ- ated, i.e. when unlabeled trimethylsilylated a- and P-methyl- glucoside were added to the trimethylsilyl derivative of the methylglucoside isolated from the reaction of [14C]sucrose with sucrose phosphorylase in aqueous methanol, the peak of radio- activity corresponded only with the a-methylglucoside peak on the gas chromatogram (see “Results”).

pH values were measured with a Radiometer glass electrode pH meter.

RESULTS

Enzymatic Xethanolysis-Sucrose phosphorylase is suffi- ciently stable in water-methanol mixtures (see below, Table II) so that the ability of methanol to act as an acceptor of the gluco- syl moiety of sucrose could be tested. The enzyme was incuba- ted in 25% methanol with uniformly labeled [14C]sucrose as substrate. The radioactive products were separated by paper chromatography and identified as glucose, fructose, and methyl- glucoside (Fig. 1). The radioactivity recorder trace was xeroxed, and the peaks were cut out and weighed. The propor- tion of the weight,s indicated that the relative amounts4 of the

4 The radioactivities of fructose, glucose, and methylglucoside reflect the amounts of these substances present at a given time after the reaction had been initiated. These data may not reflect either the rates of formation of these products or an equilibrium mixture; therefore, they are not directly comparable to the kinetic data representled in Fin. 5.

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534 Sucrose Phosphorylase: Water and Alcohols as Acceptors Vol. 247, So. 2

Fructose (

FIG. 1. Paper chromatographic identification of [W]methyl- glucoside. 0.05 ml of 0.04 M [Wlsucrose (uniformly labeled, 0.75 X lo7 cpm per Fmole) was combined with 0.05 ml of 0.04 M unlabeled sucrose, 0.15 ml of 0.05 M pyridine acetate buffer, pH 6.5, and 0.10 ml of methanol. Part of the mixture (0.05 ml) was extracted for control assays. To the remainder (0.30 ml) was added 0.05 ml of sucrose phosphorylase (500 units per ml). The solution was al- lowed to incubate at room temperature for 60 min. After this time, 0.05 ml was removed for glucose determination (Glucostat) and 10 ~1 were assayed for enzymic activity. The rest of the sample was lyophilized to dryness. The dry sample was redis- solved by adding the following solutions of unlabeled compounds: 0.1 ml of 0.1 M glucose, 0.1 ml of 0.1 M fructose, 0.5 ml of 0.2 M

a-methylglucoside, and 0.3 ml of water. A paper chromatogram was prepared as follows. Fifty microliters of the redissolved sample were spotted in the center of the origin. On one side equally spaced were spotted 5 ~1 of 0.1 M fructose standard and 5 11 of 0.1 M glucose standard; on the other side, 5~1 of 1.0 M a-

methylglucoside standard and 5~1 of 1.0 M sucrose standard were spotted. The chromatogram was developed as described under “lMethods,” the spots were detected with silver nitrate stain (15), and the radioactivity was located by means of a Tracerlab 4~ strip scanner adjusted to the 10 K setting. Radioactive dye was used in order to align the chromatogram strip with the radioactivity scan.

a-Methylglucwde

,c-Methylglucoside

I H 3 Mln

:

FIG. 2. Gas chromatographic identification of ol-[Wlmethyl- glucoside. The fractions of [Wlmethylglucoside isolated from a Dowex l-OH- column (see “Methods”) were combined and lyoph- ilized to dryness. The dry residue was redissolved to 0.1 ml with water to give a solution approximately 0.5 M with respect to CZ- methylglucoside. Ten microliters of this solution were trans- ferred to 0.1 ml of dimethylformamide; to this was added 1 ml of silylating reagent (13). The precipitate was allowed to settle, and 20~1 of the supernatant solution containing trimethylsilyl-a- methylglucoside and the trimethylsilyl derivative of the [‘4C]- methylglucoside were injected into the gas chromatographic col- umn which was fitted with an effluent stream splitter. The lower trace corresponds to the mass peaks of the gas chromatogram: the solid line is the actual recorder trace for trimethylsilyl-Lu-methyl- glucoside; the dotted line represents the relative position of tri- methylsilyl-/3-methylglucoside as determined by several separate experiments in which an admixture of the two anomers was in- jected into the column. The upper truce is the actual trace of the Nuclear-Chicago gas flow counter through which 95% of the effluent stream was diverted.

0 3 6 9 0 3 6 9 Min Mln

FIG. 3. Sucrose phosphorylase-catalyzed alcoholysis of sucrose. For each experiment, a total volume of 2.0 ml contained 0.2 ml of 1 M sucrose, 0.95 ml of 0.2 M Tris-maleate buffer, pH G.5 (the pH was readjusted if necessary), 0.37 ml of water, and 0.48 ml of the appropriate solvent. At zero time, 15 liters of sllcrose phospho- rylase (500 units per ml) were added. Aliquots of 0.4 ml were removed and boiled at 3, 6, and 9 min. One-tenth milliliter of each aliquot was assayed for glucose and fructose content using hexokinase, phosphoglucoseisomerase, and glucose-6-P dehydro- genase (5). 0, no nonaqueous solvent; n , 247; methanol (6.0 M) ;

0, 24% ethylene glycol (4.2 M); A, 24% ethanol (4.2 II). A, fructose produced; B, glucose produced.

products were fructose-methylglucoside-glucose (5:4: 1). The methylglucoside was isolated from the other components of the reaction mixture by means of column chromatography on Dowes l-OH- (16). It was then converted to the trimethylsilyl deriv- ative by the procedure described earlier (6). Authentic (Y- and P-methylglucoside were also converted to the corresponding trimethylsilyl derivatives. The gas chromatographic retention times of the trimethylsilyl derivatives of cr- and P-methylgluco- side are separated by approximately 3 min under the condit’ions described under “Methods.” Fig. 2 shows that the radioac- tivity of the methylglucoside formed via the sucrose phospho-

rylase-catalyzed methanolysis of [14C]sucrose is associated es- elusively with the a-methylglucoside derivative. This result confirms that the solvolytic reaction, as does the phoaphoro- lytic reaction, proceeds with retention of configuration at, the anomeric carbon atom of the glucosyl moiety.

Reaction with Other Alcohols-Since sucrose phosphorylase apparently transfers glucose to methanol more readily4 than to water (Fig. l), the reactivity of other hydroxylic, potential ac- ceptors was investigated. Fig. 3 depicts the rates of fructose Rnd glucose formation via enzyme-catalyzed solvolysi:: of su- crose in mixtures of water with methanol, ethanol, and et,hylene glycol. Under the same conditions, chloroethanol and mercap- toetha,nol (even at concentrations as low as 7 ‘%) completely and irreversibly inactivate the enzyme. In all the cases shown in Fig. 3A, except for water alone, the rate of formation of fructose is greater than the rate of formation of glucose. Formation of fructose in excess of glucose indicates that a glucoside was formed in addition to the hydrolytic reaction. However, t)he hydrolytic rate (i.e. rate of formation of glucose) in the presence of each alcohol is diminished relative to the rate in water alone (Fig. 3B). In the pure case of rate-limiting breakdown of an intermediate by mixed solvents (17-19) the hydrolytic rate remains constant while the over-all rate increases, reflecting the reaction of the intermediate also with the other component of the solvent. In this particular case, the diminution of the hydrolytic rate is very

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ISBUC of January 25, 1972 J. J. Mieyal, M. Simon, and R. H. Abeles 535

A

0.06

n

1 I

0 2 4

[Ethylene GI~COI] , M

Fro. 4. Depelrdence of rates on ethylene glycol concentration. A, Tris-Maleate Buffer-A, Curve F-l, v = micromoles of fructose produced per min per unit of sucrose phosphorylase; 0, Curve G-l, micromoles of glllcose per min per unit; Curve D-l, algebraic difference between Curves F-1 and G-l representing the rate of production of hydroxyethylglucoside. For each concentration of ethylene glycol, the incuba.tion mixture contained 0.21 ml of Tris- maleate buffer, 0.6 M, pH 6.5, 0.10 ml of 1 M sucrose solution, and the appropriate vohlmes of water and concentrated ethylene glycol solution to give the desired ethylene glycol concentration in a total volume of 1.26 ml. The pH of each solution was ad- justed to 6.35 & 0.05. One milliliter was transferred from each sample t,o serve as the reaction mixture; the remainder of the sam- ple was used as the zero time control. Ten microliters of enzyme solution (3.5 units) were added to each 1.0.ml sample at zero time; 0.3.ml wliquots were transferred to each of three small test tubes and boiled at 4, 8, and 12 min, respectively. Incubations were carried ollt in a constant temperature room which maintained

similar for all three alcohols, whereas the excess of fructose formed is quite different. The solvent composition (percentage of nonaqucous solvent) is the same, and the dielectric constants

of the alcohols are similar (24, 33, and 37 for ethanol, methanol, and ethylene glycol, respectively, (20)). Hence, the diminution

of the hydrolytic rate may simply reflect, an effect of the solvent on the stability of the enzyme, or it may indicate a competition of the solvent with water. In order to distinguish these possi-

bilities, the dependence of the reaction rates on the concentra- t,ions of the nonwater acceptors was further investigated.

Dependence of Rates on Acceptor Concentration--Figs. 4 to 7 represent the results of studies, each carried out in two different

buff cri;, of the dependence of the rate of enzyme-catalyzed

cleavage of sucrose on the concentrations of ethylene glycol, methanol, Irans-1,2-cyclohexanediol, and cis-1,2-cyclohexane- dial, respectively. In all cases, Part A of each figure pictures the

rates of glucose, fructose, and glucoside production (Curves G, F, and D, respectively). Curves D are the algebraic difference betwvrcn Curves F and G, i.e. the sum of the quantities of glucose and glucoside formed at any time must equal the quantity of

fructose formed. This criterion was confirmed for the case of methylglucoside formation by direct measurement of the radio- activity of the three products (Fig. I). Part B of each figure

shows the double reciprocal plots of the data of Curves D. Several features of the four figures are similar and several are

0

100 /

D-2

I I / VA I I/v I / / I

I A / / I :I

-I 0 2 4

[Ethylene Glycol]-‘,M-’

28-29”. One-tenth milliliter of each 0.3-ml aliquot was assayed for glucose and fructose content as described under “Methods.” MES Buffer-A, Curve F-2, micromoles of fructose per min per unit; l , Curve G-8, micromoles of glucose per min per unit; Curve D-d, algebraic difference between Curves F-6 and G-d representing rate of production of hydroxyethylglucoside. For each concen- tration of ethylene glycol, the incubation mixture contained 0.25 ml of 0.2 M sucrose, 0.25 ml of 1 M potassium morpholinoethane- sulfonate buffer, pH 6.5, and the appropriate proportion of water and ethylene glycol to give the desired ethylene glycol concentra- tion in a total volume of 1 ml. Ten microliters of sucrose phospho- rylase (5 units) were added to each sample at zero time. Aliquots (0.4 ml1 were boiled a.t 5 and 10 min. One-tenth milliliter of each quenchid sample was assayed for glucose and fructose content. The difference between the two quantities is taken as the amount of hydroxyethylglucoside formed. Incubations were carried out at room temperature (25-26”). B, l/v versus l/S. Double recip- rocal plots of data from Curves D-1 (0) and D-Z (0) of A.

quite different’. In all cases, the rate of hydrolysis in Tris-male-

ate buffer (G-l) is substantially greater (-&fold) than that in

MES buffer (G-W). In all cases (except cis-cyclohexanediol in Tris-maleate buffer), the variat,ion of the rate of formation of glucoside with acceptor concentration appears to approach a maximum, and the data fit fairly well to a linear double reciprocal

presentation. Hence, for the purpose of comparison, we may assume that this behavior is indicative of normal enzyme satu- ration and may estimate values of apparent maximal velocity and apparent K, from the intercepts on the ordinate and ab-

scissa, respectively, of the Lineweaver-Burk plots. Table I lists

the values of apparent K, and V mitx which have been determined for various glucosyl acceptors. In Figs. 4A to 7A, the relation- ship among the rates of over-all reaction (fructose formation

(F)), hydrolysis (glucose formation (G)), and alcoholysis (glu- coside formation (D)) change in certain cases in going from one buffer to the other with the same acceptor, and from one accep- tor to another in the same buffer. The relationship varies from

a pattern in which t’he rate of hydrolysis is essentially constant and the over-all rate and the rate of alcoholysis are parallel (Figs. 48-1, ,!?; 6A-2) to a pattern where the over-all rate is essen- tially constant and the rates of hydrolysis and alcoholysis are

inversely proportional (Figs. BA-d and 7A-1) to the case where there is no difference between the over-all rate and the rate of hydrolysis (7A-9). The data (Figs. 4 to 7) in most cases for

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536 Sucrose Phosphorylase: Water and Alcohols as Acceptors Vol. 247, so. 2

2 6

[Methanol], M -0.5 0 0.5 I.0

[Methanol]-‘, M-’

moles of fructose per min per unit; l , Curve G-Z, micromoles of glucose per min per unit; CWVC D-2, algebraic difference (F-2 minus G-2)) representing micromoles of oc-methylglucoside per min per unit. The experiments were carried out as described under Fig. 4A, “MES BufYer”, except that appropriate volumes of methanol and water were substituted for the additions of ethylene glycol and water. B, l/v versus l/S. Double reciprocal plots of the data from Curves D-1 (A) and D-2 (A) of A.

FIG. 5. Dependence of rates on methanol concentration. A, v ve~~z~s S. Tris-Maleate Buffer-A, Cure F-l, v = micromoles of fructose per min per unit; 0, Curve G-l, micromoles of glucose per min per unit; Curve D-1, algebraic difference between Curves F-f and G-f, representing the rate of production of a-methyl- glucoside. The experiments were carried out as described under Fig. 4A, “Tris-Maleate Buffer”, except that an appropriate vol- ume of concentrated methanol solution was substituted for the ethylene glycol addition. MES Buffer-A, Curve F-d, v = micro-

002 A

8 B 003

002

”

001

1000 :Y’ I/v

t--A / 500 f/A / / F-Z

-3 0 6

[Cls-1,2-Cyclohexonedlol] -I

0 0.2 0.6 IO

[CIS-1,2-Cyclohexonedlol] .M

0 04 OS [Trans-l.2-cyclohexonedial]~‘,M-’

[Trans-1,2-cyclohexonedlol], M

FIG. 6. Dependence of rates on trans.1,2-cyclohexanediol con- centration. A, v versus S. Tris-Maleate Buffer-A, Curve F-l, v = micromoles of fructose per min per unit; o , Curve G-l, micro- moles of glucose per min per unit; Curve D-l, algebraic difference (F-l minus G-l), representing micromoles of hydroxycyclohexyl- glucoside per min per unit. Experiments were carried out as de- scribed under Fig. 44, “Tris-Maleate Buffer,” substituting ap- propriate volumes of a concentrated solution of trans.1,2-cyclo- hexandiol and of water for additions of ethylene glycol solution and water. MES Buffer-A, Curve F-2, v = micromoles of fruc- tose per min per unit, l , Curve G-6, micromoles of glucose per min per unit; Curve D-2 (F-2 minus G-2), micromoles of hydroxycyclo- hexylglucoside per min per unit. For each concentration of trans- cyclohexanediol the incubation mixture contained 0.32 ml of potassium morpholinoethanesulfonate buffer, pH 6.9, 0.10 ml of 1 M sucrose solution, and the appropriate volumes of water and concentrated trans-1,2-cyclohexanediol solution to give the de- sired concentration of cyclohexanediol in a total volume of 1.26 ml. One milliliter was transferred from each sample to serve as the reaction mixture; the remainder of the sample was used as the zero time control. Ten microliters of enzyme solution (3.5 units) were added to each l.O-ml sample at zero time; 0.25-ml aliquots were boiled at 4, 8, and 12 min. Incubations were carried out at room temperature (25-26’). One-tenth milliliter of each boiled aliquot was assayed for glucose and fructose content as described under “Methods.” B, l/v versus l/S. Double reciprocal plots of the data from Curves D-l (A) and D-Z (A) of A.

FIG. 7. Dependence of rates on cis-1,2-cyclohexanediol con- centration. A, v versus S. Tris-Maleate Buffer-Curve F-1, G-l; a, micromoles of fructose per min per unit; 0, micromoles of glucose per min per unit. Experiments were carried out i;s de- scribed under Fig. 48, “Tris-Maleate Buffer,” substituting appropriate volumes of a concentrated solution of cis-I,?-cyclo- hexanediol and of water for the additions of ethylene glycol solu- tion and water. MES Buffer-A, Curve F-2, micromoles of fruc- tose per min per unit; l , Curve G-2, micromoles of glucose per min per unit; Curve D-2 (F-2 minus G-2), representing micromoles of hydroxycyclohexylglucoside per min per unit. The experiments were carried out as described under Fig. 6A, “MES Buffer,” ex- cept that addition of cis-1,2-cyclohexanediol solution was sub- stituted for addition of trans.1,2-cyclohexanediol solution. B, l/v versus l/S. Double reciprocal plot of data from CU~OC D-2 of A.

the highest concentration of acceptor deviate significantly from the regular progression of the other points on each curve. We have observed in experiments where the concentration ranges were extended, that this downward trend in the rates continues for concentrations of acceptors higher than those shown on the graphs. With other enzyme-substrate interactions, such devia- tions are indicative of substrate inhibition, and there are many interpretations of how this type of inhibition could arise (21). In the present case, one interpretation which must be considered is that the diminution of the rates may reflect solvent-induced

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TABLE I

Relative reactivities of glucosyl acceptors

TABLE II

Stability of sucrose phosphorylase in various mixtures

Pbb Fructoseb Sorboseb Ethylene glycolc

Irans-l,Z-Cyclo- hexnnediolc

Methanolc

cis-1,2-Cyclo- hexanediolc

Ethanold Watere

-I-

pparent K, Apparent vm.x

‘M

0.002 0.013 0.130 0.87 2.7 0.40 0.27 2.3 2.1 0.39

pmoles/ min/unit 1.40 0.91 0.62 0.053 0.046 0.023 0.014 0.038 0.011 0.0046

700 TM 70 TM 4.8 TM 0.061 TM 0.017 MES 0.057 TM 0.052 MES 0.017 TM 0.005 MES 0.015 MES

“2.1" 0.004 0.002 TM "20" 0.023 0.001 TM “20" 0.005 0.00025 MES

a TM = Tris-maleate; MES = sodium morpholinoethane sul- trans.1,2-

Eonate. Cyclohcxanediold b Values taken from Silverstein et al. (5). c Values estimated from intercepts of Figs. 4B, 5B, BB, and 7B. d Single experiment (Fig. 3). “K,” is simply taken as one-half

the concentration of ethanol used. e The upper limit of the K, for water as 20 M was estimated as

follows. Since ethylene glycol-water mixtures of up to 25y0 ethylene glycol in most cases do not significantly affect the rate of glucose production, it is assumed that 75y0 water is still a “saturat,ing” concentration for the enzyme; hence, one-half of this value may represent an upper limit for “.Gwster,” i.e. 0.75 X 56 M (pure water) f 2 M 20 M.

c&l, 2- Cyclohexanediold

Q For each nonaqueous component, a separate control experi- ment was performed in which the incubation mixture and condi- tions were the same (see details in Footnotes 6, c, and d below) except that the nonaqueous component was replaced by an equal amount of water. The assayed activity for each control was designated 100%. During a period of 10 min, aliquots were with- drawn from the incubation mixtures containing sucrose, phos- phate, and enzyme, and quenched, appropriately diluted and each assayed for glucose-l-P content (Column 4) by the procedures described under “Methods.” After this time period, a microliter quantity of the reaction mixture was diluted into standard assay mix (see “Methods”) in order to assay the enzyme for residual phosphorylytic activity (Column 3).

enzyme denaturation. Some evidence pertinent to evaluating such an interpretation is presented in Table II. Solvent effects were determined in two ways. (a) The rate of phosphorolysis was measured in the mixture of nonaqueous component and water (in situ activity assay). (b) The enzyme was exposed to the solvent mixture for a given period; then small aliquots were transferred to the standard assay system so that the phospho- rolytic rate was measured in an essentially aqueous environment’ (residual activity assay). With acetonitrile and dioxane, two substances which cannot act as glucosyl acceptors, the loss of residual activity even at low nonaqueous solvent concentrations closely parallels the loss of in situ activity. These results indi- cate that the enzyme is partially and irreversibly denatured in these solvent mixtures. On the contrary, with the other compounds, the residual activity is unaffected until very high concentrations of nonaqueous species are present (e.g. meth- anol, 4079, whereas the in situ rate of phosphorolysis drops off rapidly with increasing acceptor concentration. The dispar- ity between the apparent stability (residual activity) of su- crose phosphorylase in alcoholic solutions and its loss of in situ phosphorolytic activity may reflect a reversible partial denatu- ration, which is overcome when the enzyme is diluted into the assay mixture. However, the diminution of the phosphorolytic rate by the alcohols could be due to a competition with phos- phate for the acceptor-binding site. Hence, the actual patterns of inhibition by the alcohols were examined.

Inhibition of Sucrose Phosphorylase by Alcohols-Fig. 8, A and B, shows that ethylene glycol is purely noncompetitive with both sucrose and phosphate (i.e. it does not affect the K, values for

Nonaqueous component

None5 Acetonitrileb

Dioxaneb

Methanolc

Ethylene glycolb

‘ercentage bq volume or

oncentration

10 20 30 10 20 30 10 20 30 35 40 10 20 30

0.5 M 1.0 2.0 1.0 M

Relative residual Relative xtivity after diluting ctivity in the out the nonaqueous mixture

component (in situ)

100 73 11 0

77 62

9 97

104 96

9 100 106 100

100

100

-

100 69 12 0

76 51 19 88 66 38 21

67 38 19 82 54 32 57

b The data for residual activity and in situ phosphorolysis were obtained from reaction mixtures containing in a total volume of 2 ml: 0.2 ml of 0.5 M sucrose, 0.2 ml of 0.5 M potassium phosphate, pH 7, 0.5 ml of 1 M dimethylglutarate, pH 7, and the appropriate volumes of water and nonaqueous solvent. Approximately 2.5 units of sucrose phosphorylase were added at zero time.

c Each incubation mixture contained in a total volume of 2.0 ml: 1.0 ml of 1 M potassium phosphate buffer, pH 7,0.25 ml of 0.4 hf sucrose, and the appropriate volume of methanol and water to give the desired volume percentage of methanol. Approximately 3 units of sucrose phosphorylase were added at zero time.

d For the determination of residual activity, or in situ phos- phorolytic activity, or both, each 1 ml of solution contained 0.1 ml of 0.5 M sucrose, 0.25 ml of 1 M morpholinoethane sulfonate, pH 6.5, 0.1 ml of 1 M potassium phosphate, pH 6.5, appropriate vol- umes of a concentrated solution of cis- or trans-diol and of water, and -6 units of sucrose phosphorylase.

either substrate). The percentage of activity of the enzyme in the presence of ethylene glycol for all concentrations of sucrose and phosphate remained constant, (38%) and is identical with that determined for the in situ activity of the enzyme in 20% ethylene glycol under the conditions listed in Table II. The inhibitor-binding constant K; for ethylene glycol calculated from the data of both Fig. 8, A and B, is 2.5 M and is approximately equal to the apparent K, determined for ethylene glycol in the sa,me buffer (2.7 M, Table I).

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538 Xucrose Phosphorylase: Water and Alcohols as Acceptors Vol. 247, No. 2

A 8 --

-400 -200 0 200 400

[Sucrose]-‘, M-’

r 8 8

-400 -200 0 200 400

[Phosphate]-‘,M-’

FIG. 8. Inhibition of sucrose phosphorylase by ethylene glycol. A, [Sucrose] varied: two series of three incubat,ion mixtures were prepared. The mixtures in one of the series contained no ethylene glycol (0) ; the mixtures in the other series each were 20% (3.6 M)

with respect to ethylene glycol (A). The sucrose concentrations for the three mixtures in both series were 0.0025, 0.005, and 0.1 M;

the phosphate concentration was constant. All of the incubation mixtures contained 0.25 ml of 0.5 M potassium phosphate, pH 7, 0.5 ml of 1 M potassium morpholinoethanesulfonate, pH 6.9, and the appropriate volumes of concentrated sucrose solution, ethyl- ene glycol, and water to give the desired concentration of ethylene glycol (0 or 20%~) and sucrose (0.0025 to 0.1 M) in a volume of 2.0 ml, pH 6.9. Three-tenths milliliter was withdrawn from each mixture to serve as zero time control. Then 10~1 of enzyme (1 unit) were added to each, and 0.3.ml aliquots boiled at 3, 6, and 9 min. One-tenth milliliter of each boiled aliquot was assayed for glucose-l-P content as described under “Methods.” l/v repre- sents [micromoles of glucose-l-P per min per unit of enzyme]-‘. B, [Phosphate] varied: two series of three incubation mixtures were prepared: no ethylene glycol (0) ; 20% (3.6 M) ethylene glycol (A). The phosphate coucentrations for the three mixtures in both series were 0.0025, 0.005, 0.1 M; the sucrose concentration was constant. All of the incubation mixtures contained 0.4 ml of 0.4 M

sucrose solution, 0.5 ml of 1 M potassium morpholinoethanesul- fonate, pH 6.9, and the appropriate volumes of concent,rated phos- phate solution, ethylene glycol, and water to give the desired concentration of ethylene glycol (0 or 20%) and phosphate (0.0025 to 0.1 M) in a volume of 2.0 ml, pH 6.9. Three-tenths milliliter was withdrawn from each mixture to serve as zero time control for the assays. Then, 10~1 of enzyme (1 unit) were added to each, and O&ml aliquots boiled at 3, 6, and 9 min. One-tenth ml of each boiled aliquot was assayed for glucose-l-P content as described under ‘Vethods.” l/v represents [micromoles of glucose-l-P per min per unit of enzyme]-I.

-400 -200 0 200 400

[Sucrose] -I

, M-’

-400 -200 0 200 400

[Phosphate]-‘, M-I

FIG. 9. Inhibition of sucrose phosphorylase by trans-1,2-cy- clohexanediol. A, [Sucrose] va.ried: no cyclohexanediol (O ) ; 1 M trams-1,2-cyclohexanediol (A). Experiments were carried out as described under Fig. 88, substituting appropriate volumes of a concentrated solution of trans.cyclohexanediol and of water for additions of ethylene glycol and water. B, [Phosphat,e] varied: no cyclohexanediol (0); 0.5 M trans-1,2-cyclohexanediol (m) 1.0 M trans-1,2-cyclohexanediol (A). Three series of experiments were carried out as described under Fig. SB, substituting appropri- ate volumes of a concentrated solution of trans.cyclohexanediol and of water for additions of ethylene glycol and water.

trans-l ,Z-Cyclohexanediol is purely noncompetitive with su crose (Fig. 9A) and the percentage of activity for all concenira- tions of sucrose (56%) is the same as that measured for the in : itu activity of the enzyme in 1 M trans.cyclohexanediol under the conditions of Table II (54%). Ki calculated from these data is 1.4 M, much higher than the apparent K, for trans-cyclo- hexanediol in MES buffer (0.27 M, Table I). Phosphate and kuns-cyclohexanediol are competitive (Fig. 9B). The value of Ki calculated from these data for trans-cyclohexanediol (0.25 M) is very similar to its apparent K, in MES buffer (0.27 ‘II, Table I). It should be noted that tram-1,2-cyclohexanediol is com- petitive with Pi but does not diminish the rate of hydrolysis (Fig. 6A-2).

The inhibition patterns with cis-1 ,2-cyclohexanediol for both sucrose (Fig. 1OA) and phosphate (Fig. 1OB) are indicative of mixed type inhibition. Values of Ki estimated from the slopes of the graphs, 0.4 RI (Fig. 1OA) and 0.6 M (Fig. 1OB) are similar

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Issue of January 25, 1972 J. J. Mieyal, M. Simon, and R. H. Abeles 539

FIG. 10. Inhibition of sucrose phosphorylase by cis-1,2-cyclo- hexanediol. A, [Sucrose] varied. These experiments were car- ried out via a modification of the standard continuous assay system for sucrose phosphorylase (see “Methods”). l , no cyclo- hexanediol; A, 1 M cyclohexanediol. For determination of each value of v (micromoles of glucose-l-P per min per unit of enzyme), the following additions were made to a l-ml cuvette: 0.09 ml of 6 X 10m3 M MgS04 solution containing 2~1 of mercaptoethanol per 0.09 ml of solution, 0.16 ml of 0.5 M potassium phosphate, 0.07 ml of NADP solution (4 mg per ml), and the appropriate volumes of concentrated sllcrose solution, concentrated cis-1,2-cyclohexane- diol solution, and water to give the desired concentration of su- crose (0.0025,0.005,0.1 M) and the desired concentration of cis-cy- clohexanediol (0 or 1 M) in a total volume of 0.8 ml. Then, 2 ~1 of glucose-6-P dehydrogenase (5 mg per ml), 5~1 of phosphoglucomu- tase (10 mg per ml), and 2~1 of dilute sucrose phosphorylase (0.005 units) were added, the cuvette shaken, placed in the spectropho- tometer at 30” and continuous formation of NADPH monitored at 340 nm for at least 10 min. Linear slopes were obtained from which the rates (micromoles of glucose-l-P per min per unit) were calculated. B, [Phosphate] varied. These experiments were car- ried out as described under A, except for the additions of sucrose and phosphate. In this case, 0.08 ml of 1 M sucrose solution was added to all assay mixtures, and appropriate volumes of concen- trated phosphate solutions and of water were added to give the desired concentrations of phosphate (0.0025, 0.005, 0.1 M) in the 0.8 ml of each solution.

-400 -200 0 200 400

[Sucrose] -I

, M-’

6

4 --

-400 -200 0 200 400

[Phosphate]-‘, M-’

Glucose-l-P Glucose

Fructose Sucrose Chromatographic

1 Origin Methylglucoside

Marker

Dye

Marker

Dye

FIG. 11. Simultaneous formation of glucose-l-P and a-methyl- the sample along with standards for sucrose, cY-methylglucoside, glucoside. One-tenth milliliter of 0.04 M [l%]sucrose (uniformly and glucose-l-P were spotted on Whatman No. 3Mh1 chromatog- labeled, 0.86 X lo7 cpm per micromole) was combined with 0.04 raphy paper. The chromatogram was developed descendingly ml of 0.1 M potassium phosphate, pH 7, 0.1 ml of methanol, and (solvent allowed to drip off serrated edge) for 18 hours in ethyl 0.16 ml of pyridine acetate, pH 5.5; resultant pH 6.3. Sucrose acetate-pyridine-water (12:5:4). The sugar spots were detected phosphorylase was added and the reaction was allowed to proceed with silver nitrate stain (15), and glucose-l-P was also located at room temperature for 15 min. At the end of this period an with molybdate-phosphate stain (15). The radioactivity was aliquot was assayed for enzymic activity: 0.16 unit was present located by means of a Tracerlab 4~ strip scanner. The lower trace in the reaction mixture. The solution was frozen, lyophilized to was recorded at a setting of 10 K, the upper trace at a 1 K setting. dryness, and then redissolved by adding: 0.03 ml of 0.5 M sucrose, The relative radioactivities were determined by relating the areas 0.06 ml of 1 M a-methylglucoside, 0.06 ml of 1 M glucose-l-P, 1~1 of individual peaks in each trace to the area of the peak for the of 0.1 M glllcose, and 1 pl of 0.1 M fructose. Twenty microliters of radioactive dye marker in the respective trace.

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540 Xucrose PhosphoTylase: Water and Alcohols as Acceptors Vol. 247, No. 2

t’o the estimated apparent K, for cis-cyclohexanediol (0.39 M,

Table I). Simultaneous Phosphorolysis and Solvolysis--Because of the

great difference between the rates of solvolysis and phosphoroly- sis, small amounts of solvolytic products are not discernible by means of spectrophotometric assay from a difference between the amounts of glucose-l-l’ and fructose produced (i.e. a differ- ence of 1 to 471 between the absorbance readings for NADPH produced by oxidation of the glucose-6-P from glucose-l-P and fructose is Jvithin the limit of errors). One method of learning whether glucose or other glucosides are formed simul- taneously with glucose-l-P when phosphate is present in the reaction mixture is to use [14C]sucrose and separate and analyze the products according to relat#ive radioactivit’y (Fig. 11). The relative areas of the peaks in Fig. I1 yield the following data: approximately 407, (1.6 pmoles) of the sucrose present initially had been converted to products. This means that the concen- trations of sucrose and Pi were reduced from 0.01 to -0.006 M,

or from about 5 times their K, values to about 3 times K, (5). Hence, it may be assumed that the rates of formation of prod- ucts did not change greatly during the time of incubation. Approximately 1.5 pmoles of glucose-l-P and 0.06 pmole of methylglucoside were formed. The rate of mcthylglucoside formation est’imated from these data is -0.027 pmole per min per unit of enzyme. This rate is very similar to that observed in Tris-maleate buffer in the absence of Pi (Fig. 58-1, Curve D-i (4.2 M methanol)).” This result suggests that phosphate does not interfere with methanolysis.

DISCUSSION

In addition t’o water, a number of alcohols can function as glucosyl acceptors in reactions catalyzed by sucrose phosphoryl- ase. The rates of reaction of these alcohols (Table I) are con- siderably slower than those of the acceptor sugars or Pi and are more comparable to the rate of the hydrolytic reaction cata- lyzed by sucrose phosphorylase. The slowness itself suggests that glucosyl enzyme breakdown must be the rate-limiting step of these react#ions. Consistent with this interpretation is the observation t,hat the rates of hydrolysis of sucrose, glucose-l-P and glucose l-fluoride are identical (5, 13), although these gluco- syl donors are quite different with respect to the leaving aglu- cone. For the purpose of discussion it will be convenient to divide glucosyl acceptors into two groups: good acceptors (Pi, sugars) and poor acceptors (HsO, alcohols). ‘v,,, for the least react’ive good acceptor is 12 times that of the best poor acceptor (Table I). The stereochemical course of the reaction was in- vestigat#ed for one of the poor acceptors, methanol. It was found t,o be the same as that for the good acceptors, i.e. reten- tion of configuration at the anomeric carbon atom of the gluco- syl moiety. This result suggests that the fast and slow reactions proceed by the same mechanism.

It should also be noted that on a molar basis all of the alcohols tested (except cis-cyclohexanol in Tris-maleate buffer) react faster than water. For instance, in 2 M ethylene glycol (11% by volume) 2 to 3 times as much hydroxyethyl glucoside is formed as glucose (Fig. 4/l), indicating that ethylene glycol is 50 to 75 times as reactive as water (49 M, 89%). It appears to

5 These experiments were carried out in pyridine acetate buffer to facilitate subsequent chromatographic procedures. In sepa- rate experiments it was shown that the rate of hydrolysis in that buffer is equivalent to that in Tris-maleate buffer.

us unlikely that the differences in rates are entirely due to differ- ences in nucleophilicity. The data in Table I show that the

V max for ethylene glycol, methanol, and 1,2-trans-cyclohexane- diol, is very similar to that of water, which supports the notion that their intrinsic reactivities are very similar. The higher reactivity of the alcohols relative to that of water, observed when the alcohols are at low concentrations, can be best ex- plained by the presence of a specific binding site for the alcohols. The question then arises whether the poor acceptors bind at the same site as good acceptors, or whether they bind at a separate site which may or may not coincide with the site at which water reacts with the glucosyl enzyme. For cases in which addition of alcohol does not reduce the rate of glucose formation it can be concluded that the alcohol and water do not react at the same site. For the reactions with alcohols (poor acceptors) forma- tion of glucosyl enzyme cannot be rate limiting; therefore, if the alcohol does not interfere directly with water it is expected that the rate of glucose formation remain constant while the rate of glucoside formation increases.

Examples in which constant glucose formation is observed in the presence of a second acceptor are the reactions with ethylene glycol (Fig. 4$-j,%) and with trans-cyclohexanediol in MES buffer (Fig. 6LZ). Glucoside formation appears to follow saturation kinetics. The asymptotic nature of the concentra- tion dependence cannot be ascribed to solvent effects on enzyme reactivity because the rate of hydrolysis does not fall concur- rently.6 transCyclohexanedio1 is a competitive inhibitor of phosphate (Fig. 9B), and its K; (0.25 M) is very similar to its apparent K, (0.27) calculated from the data of Fig. 6B-2. It is noncompetitive with respect to sucrose, typical of the inhibi- tion pattern expected for an inhibitor of the second substrate (acceptor) in a ping-pong reaction (19). The results with trans- 1,2-cyclohexanediol are compatible with the postulate that the diol binds at the acceptor site and that water does not react at that site. There may be a separate binding site for water or water may react without binding to the glucosyl enzyme. An argument against the latter possibility is the observation that

even at 25 to 30% ethylene glycol the hydrolysis rate is not appreciably reduced, although the concentration of water is dc- creased. This insensitivity of the reaction to water concentra- tion suggests the existence of a water-binding site which is still saturated at 40 M H20.

Ethylene glycol, although it does not reduce the hydrolytic rate, is noncompetitive with both sucrose and Pi (Fig. 8, A and B). One possible model which Tvould be consistent with these results, and would not entail invoking a unique binding site for every acceptor, is that the diol ethylene glycol can bind in a multitude of orientations in the site which accommodates phos- phate, fructose, and other sugars. Such binding would explain the noncompetitive inhibition with sucrose. The additional requirement in order to explain noncompetitive inhibition with phosphate is that the acceptor-binding site is large enough to accommodate both phosphate (aligned for reaction) and ethylene

6 The data suggest that solvent effects only become important at high solvent concentrations. In cases where the hydrolytic rate is constant it remains constant over the same solvent concen- tration range for which the l/v versus l/S plot is linear. Deviation from linearity in these plots corresponds to solvent concentration at which the hydrolytic rate also decreases. Furthermore the extrapolated V,,, for the reaction with alcohols, which ignores the points which deviate from linearity, is very similar to that found for the hydrolytic rate in aqueous solvent.

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glycol (bound nonproductively). Such a model can also explain why the amount of inhibition of glucose-l-P formation by trans- 1,2+yclohexanediol (which presumably occupies the entire acceptor site and is thus competitive with phosphate) is not equalled by the amount of hydroxycyclohexylglucoside formed. Again, the 2-carbon adjacent dihydroxy region of the molecule could occupy many of the orientations of 2-carbon portions of the molecules of the better polyhydroxy sugar acceptors, and yet be bound in a nonproductive manner.

Reduction of the hydrolytic rate (i.e. glucose formation) by added acceptors is more difficult to interpret. It could be due to: (a) competition between acceptor and Hz0 at a common binding site; (b) the acceptor could exert non- or uncompetitive effects. It should be noted that none of the acceptors produces irreversible inhibition (Table II) ; (c) the acceptor could bind at the glucose-binding site (i.e. competition with the glucosyl douor) in addition to binding at the site at which it reacts; (d) glucosyl enzyme formation could become rate limiting. It seems that this hypothesis can be excluded in all cases since the ac- ceptors used are ‘Lpoor” substrates, and the total reaction rate including hydrolysis falls far below that observed with good acceptors (Table I).

Acceptors which apparently decreased glucose formation are methanol (Fig. 5A-I ,.2), cis-1,2-cyclohexanediol (Fig. 7A-I ,d), and trans-1,2-cyclohexanediol in Tris-maleate buffer (Fig. 6 A-1). In the reaction with methanol, glucose formation de- creases and there is a concomitant increase in both methylgluco- side formation and the over-all rate. The increase in methyl- glucoside formation appears to obey linear saturation kinetics as indicated in Fig. 5B. Furthermore, MES buffer reduces the

vm, by the same factor as the hydrolytic rate is reduced, but has no effect on K, for methanol. This relationship is to be compared with the reaction of ethylene glycol, where MES had no effect on V,,, but K, was reduced a-fold, and where it was

/ PH 0”

3-a-D-GIucopyronosyI-L- Arobinose

concluded that ethylene glycol binds at the acceptor site. These facts taken together suggest that methanol does not react at the acceptor site but at a separate site, the site at which water reacts. This interpretation is supported by two other observations. The rate of phosphorolysis at saturating concentrations of Pi is not reduced by MES; the presence of Pi does not significantly reduce methanolysis (Fig. 11). In connection with this consid- eration, it is interesting that the methanolysis of acetylphenyl- alanine methyl ester by chymotrypsin yielded data which Bender and Glasson (22) interpreted as indicative of methanol-water competition for a specific binding site. An alternative explana- tion in the present case is that methanol binds at a site different from both the acceptor site and the Hz0 site and reduces the hydrolytic rate through a noncompetitive or uncompetitive effect. This possibility cannot be totally excluded. The data of Table II show that methanol may exert such an effect on the phosphorolytic rate. However, it is also possible that methanol may bind at both the Hz0 site and the acceptor site, reacting only at the Hz0 site, but exerting inhibitory effects at the other.

The reactivity of cis-1,2-cyclohexanediol in XES buffer re- sembles that of methanol in that there is a concomit,ant decrease in glucose formation with increase in glucoside production; but the over-all rate apparently does not change. These results, as well as the reaction of cis-cyclohexanediol in Tris-maleate buffer and of frans-1,2-cyclohexanediol in Tris-maleate buffer, are subject to several interpretations. It is possible that in these cases the acceptor binds at the Hz0 site and competes with water. For cis-cyclohexanediol in MES buffer and Irans-cyclohexane- diol in Tris-maleate buffer, the extrapolated I’,,, for glucoside formation is equal to the hydrolytic rate in the absence of the acceptor. The over-all rate, i.e. glucoside plus glucose formed, remains constant, indicating that these diols can totally repress the hydrolytic rate. This effect suggests interaction at the water site. If this conclusion is accepted as well as the previous

H H HH* H

H H

H H

HO H

cis-1,2-Cyclohexanediol

H CH20H

HO 0

H

HO

m O-Enzyme

HO H H

P-D-Glucopyranosyl -Enzyme trans - 1,2-Cyclohexanediol

FIG. 12. Structures of some reactants in sucrose phosphorylase reactions.

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542 Sucrose Phosphorylase: Water and Alcohols as Acceptors Vol. 247, r\;o. 2

argnments, it follows that trans-1,2-cyclohexanediol binds at the Hz0 site in Tris-maleate buffer and at the acceptor site in MES buffer. Xternatively, it could be postulated that in all or some of these cases, the alcohols bind at the acceptor site and inhibit the hydrolytic reaction in a non- or uncompetitive man- ner. If this is true, cis-cyclohexanediol in Tris-maleate buffer binds overwhelmingly nonproductively since no glucoside is formed. In this particular case a third alternative which can- not be ignored is that cis-cyclohexanediol might bind at the glucosyl-binding site, thereby inhibiting all reactions.

It may be concluded that two independent sites are available on sucrose phosphorylase for the reaction of acceptors with the glucosyl enzyme. One site is designated as the Hz0 site and the other as the acceptor site. Probably all of the good acceptors react at the acceptor site as well as poor acceptors such as trans- 1,2-cyclohexanediol and probably ethylene glycol. Methanol, a poor acceptor, probably reacts at the H20 site. The site of interaction of some other acceptors is uncertain. It appears that a necessary, although possibly not a sufficient condition, for reactivity at the acceptor site is two adjacent hydroxyl groups on adjacent carbon atoms as proposed by Gottschalk (8). Fig. 12 shows the structures of 3-c-D-glucopyranosyl I,-arabino- pyranose (the disaccharide which prompted Gottschalk’s con-

elusion), cis- and trans.1 ,2-cyclohexanediol, and B-D-glUCOpy-

ranosyl enzyme. The dotted line shows the only region of homology of all the acceptors reported prior to this publication. The proposal that the hydroxyl group adjacent to the glycosidic oxygen atom must be “cis-disposed and codirectional” to the hydroxyl group on C-2 of the glucosyl moiety may not be appli- cable since frans-1,2-cyclohexanediol appears to react at the

acceptor site.

REFERENCES

1. DOUDOROFF, M., KAPLIIN, N., AND HASSID, W. Z. (1943) J. Biol. Chem., 148, 67.

2. COHN, M. (1949) J. Biol. Chem., 180, 771. 3. FITTING, C., AND DOUDOROFF, M. (1952) J. Biol. Chem., 199,

4.

5.

6.

7. 8. 9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

153. DOUDOROFF, M., HSSSID, W. Z., AND BARKER, H. A. (1947) J.

Biol. Chem., 168, 733. SILVERSTEIN, R., VOET, J., REED, D., AND ALII”LES, R. H.

(1967) J. Biol. Chem., 242, 1338. VOET, J. G., AND ABELES, R. H. (1970) J. Biol. Chem., 246,

1020. DETOMA, F., AND ABELES, R. H. (1970) Fed. Proc., 29, 461. GOTTSCHALB, A. (1950) Advan. Carbohyd. Chem., 6, 49. WEIMBERG, R., AND DOUDOROFF, M. (1954) J. Bacterial., 68,

381. COHN, M. (1961) in P. D. BOYER, H. A. LARDY, AND K. MYR-

B.&K (Editors), The enzymes, Vol. 5, Ed. 2, p. 179, Academic Press, New York.

DOUDOROFF, M. (1961) in P. D. BOYER, H. A. LAXDY, AND K. MYRB;~CK (Editors), The enzymes, Vol. 5, Ed. 2, p. 229, Academic Press, New York.

GLASER, L. (1964).in M. FLORKIN .~ND E. H. STOTZ (Editors), Comvrehenisve biochemistru. VoT. 15. w. 93. American Elsevier Pubfishing Co., New York: ‘- ’

GOLD, A. M., AND OSBEIZ, M. P. (1971) Biochem. Biophys. Res. Commun., 42, 469.

DuBors, M., GILLES, K. A., AND ~~~~~~~~~~ J. K. (1956) Anal. Chem., 28, 350.

SMITH, I. (1960) Chromotographic and electrophoretic techniques, Vol. 1, Ed. 2, p. 248, Interscience Publications, Inc., New York.

ROSEMAN, S., ARELES, R. H., AND DORFMAN, A. (1952) ~lrch. Biochem. Biophys., 36, 232.

CAPLO~, M., AND JIENCKS, W. P. (1964) J. Biol. Chem., 239, 1640.

GREENZ~ID, P., AND JEKCKS, W. P. (1971) Biochemistry, 10, 1210.

JENCKS, W. P. (1969) Catalysis in chemistry and enzywLoZogy, McGraw-Hill Book Co., New York.

WEAST, R. C. (Editor) (1970) Handbook of chemistry and physics, Ed. 51, p. E-61, The Chemical Rubber Co., Cleveland.

DIXON, M., AND WI:UU, E. C. (1964) Enzymes, p. 75, Academic Press, New York.

BENDER. M. L.. .~XD GLASSON. W. A. (1960) J. Amer. Chem,. Sot., 82, 3336:

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John J. Mieyal, Marcia Simon and Robert H. AbelesWATER AND OTHER ALCOHOLS

Mechanism of Action of Sucrose Phosphorylase: III. THE REACTION WITH

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