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THE JOURNAL OF BIOLOGICAL CHE~~TRY Vol.245, No. 5,Issueof March 10,~~. 1020-1031, 1970

Printed in U.S.A.

The Mechanism of Action of Sucrose Phosphorylase

ISOLATION AND PROPERTIES OF A ,&LINKED COVALENT GLUCOSE-ENZYME COMPLEX*

(Received for publication, October 9, 1969)

J.G. VOET~AND R.H. ABELES

From the Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02154

SUMMARY

When sucrose phosphorylase reacts with sucrose, a covalent glucose-enzyme complex can be isolated. In one case, the complex was isolated by rapidly denaturing the enzyme under acidic conditions. In the other case, the complex was isolated by chemically modifying the protein with NaI04 after addition of the substrate. The NaIOd-modified complex maintains the ability to transfer the glucosyl group to acceptors with an acceptor specificity that resembles the native enzyme. The rate of reaction is approximately J- that of the native enzyme. Pepsin digestion of both 20,000 types of complexes produces positively charged, glucose- containing fragments, presumably peptides. The linkage between glucose and peptides as well as between glucose and enzyme is extremely base labile. For the glucosyl- peptides, t+ of glucose release at pH 6.0 is 80 min at 25’. When glucose is released from glucosyl peptides in ‘75% methanol, only glucose and no methyl glucoside is formed. It was concluded that glucose must be linked to the peptide through an oxygen atom contributed by the peptide and that the C-l glucose oxygen bond was not broken during solvolysis. The glucose released from the peptides has the p configuration. Therefore, formation of the glucose-enzyme complex proceeds with inversion of configuration at the C-l atom of the glucosyl moiety of sucrose. This fact, together with the isolation of a glucose-enzyme complex, provides support for the hypothesis that sucrose phosphorylase func- tions through a double replacement mechanism with intermediate formation of a P-linked glucose-enzyme complex.

Sucrose phosphorylase (disaccharide glucosyltransferase EC 2.4.1.7) catalyzes a number of glucosyl transfer reactions (l-4) ;

for example, sucrose + Pi = ar-D-glucose-l-P + n-fructose. In addition to Pi and n-fructose, L-sorbose, u-xylulose, L-arabinose,

* Publication 696 from the Graduate Department of Biochemis- try, Brandeis University, Waltham, Massachusetts 02154. This work was supported by Grant GB 5704 from the National Science Foundation and United States Public Health Service Training Grant GM 212.

$ Recipient of United States Public Health Service Predoctoral Fellowship 5-Fl-GM 29852. Present address, Department of Bio- chemistry, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104.

L-arabinulose, AsO,=, and Hz0 can also serve as glucosyl acceptors. The reaction with AsOp or Hz0 leads to the formation of glucose and is irreversible. The kinetics of the reaction (5) and isotope exchange experiments (4,6) are consistent with the intermediate participation of a glucosyl-enzyme. In general, con- vincing evidence for the existence of an enzyme-substrate complex is its direct isolation. Isolation of a complex also facilitates the identification of the functional groups involved, and therefore provides considerable evidence towards the understanding of the mechanism of action. In transfer reactions, it should be possible to isolate an intermediate enzyme-substrate complex if the substrate is added in the absence of acceptor. Unfortunately, this is not possible for sucrose phosphorylase since water can function as an acceptor. From kinetic data it can be calculated that t+ of hydrolysis of the glucose-enzyme complex in the presence of water is less than 2 sec. If a complex is to be isolated, it must be denatured rapidly so that it is not consumed by reaction with acceptors, or, alternatively, it must be chemically modified so as to reduce its rate of reaction with acceptors. Previously, the isolation of a denatured, insoluble glucose-enzyme was achieved by rapidly adding the enzyme to boiling methanol (5) in the presence of substrate. However, it was not possible with that complex to conclusively demonstrate the presence of a covalent glucose-protein bond. We have now developed more suitable procedures for the isolation of the complex. One procedure involves precipitation at acid pH after addition of the substrate. In the other, the enzyme is chemically modified after addition of substrate. This modification reduces the rate of reaction with acceptor 20,000-fold. The procedure for isolating these complexes as well as their properties is reported here.

The reaction catalyzed by sucrose phsophorylase as now en- visioned is a double replacement reacti0n.l It was pointed out by Koshland (7) that in reactions of this type, two inversions of configuration would take place at the substrate atom at which bond breakage occurs. Since the glycosidic linkage in sucrose has the a! configuration, we would expect a p configuration in the glucose-enzyme linkage. The availability of a glucose-enzyme complex enabled us to determine the configuration of the glucose-enzyme linkage and to test this prediction. To the best of our knowledge, this is the first example in which the stereochemical course of an enzymic double replacement reaction has been experimentally determined.

1 Previously, the term “double displacement mechanism” has been used (7). We prefer “replacement” since it has no mechanis- tic implications.

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Issue of March 10, 1970 J. G. Voet and R. H. Abeles

MATERIALS AND METHODS

Analytical Procedures-Sucrose phosphorylase was assayed for enzymatic activity as described previously (5).

a-n-Glucose-l-P was assayed spectophotometrically (5, 8). Glucose was determined with the Glucostat (Worthington). Su- crose and a-methyl glucoside were determined with a nonspecific carbohydrate assay involving the reaction with phenol and sul- furic acid (9). Sucrose was also determined enzymatically using sucrose phosphorylase coupled with phosphoglucomutase and glucose-6-P dehydrogenase. The incubation mixture contained 0.03 to 0.15 pmoles of sucrose, 1 unit of sucrose phosphorylase in a total volume of 0.25 ml of 0.03 M phosphate buffer, pH 7.0. After 10 min at room temperature, the mixture was made 0.075 M in Tris-chloride buffer (pH 8.0), 6 X 10m4 M in MgS04, 5 X 10V4 M

in TPN+, 5 pg per ml in glucose-6-P dehydrogenase, and 50 pg per ml in phosphoglucomutase. The final volume was 0.5 ml. After 30 min, the absorption at 340 rnp was determined and used to calculate the sucrose concentration. Glucose-6-P was determined enzymatically with glucose-6-P dehydrogenase (8). Sug- ars were detected on paper by staining either with silver nitrate (10) or benzidine (11). Concentrations of soluble proteins were determined by ultraviolet absorption (12) and by the method of Lowry et al. (13). Insoluble protein was assayed by the modified method of Lowry et al. (13).

Determination of Radioactivity-Radioactivity was determined by liquid scintillation counting as previously described (5). Ra- dioactive compounds were located on paper with the aid of a Tracerlab windowless 4?r scanner.

Radioactive Xubstrates-Glucose-l-32P was synthesized enzymatically from sucrose and 32P i with sucrose phosphorylase. The reaction mixture contained about 90 PCi of 32Pi, 10 pmoles of Pi, 20 pmoles of sucrose, and 8 units of sucrose phosphorylase in a volume of 0.2 ml. The pH of the mixture was 7. The reaction was allowed to proceed for 45 min. The mixture was then streaked onto water-washed Whatman No. 3MM paper in a lo- cm streak and subjected to descending chromatography using isopropyl alcohol-Hz0 (160:40) as the developing solvent. This procedure removes uncharged sugars. The solvent was allowed to drip off the paper. The paper was dried and the radioactive peak at the origin was cut out and sewn onto another sheet of water-washed Whatman No. 3MM paper. Marker spots of glucose-l-P and Pi were placed at either side of the strip. The chromatogram was then developed with acetone-30% acetic acid (1:l) for 16 hours (ascending). Glucose-l-P was located by staining with benzidine and radioactivity was determined with the aid of a 41r scanner. The only radioactive peak corresponded to glucose-l-P. The yield was 10.3 pmoles. The specific radioactivity of the glucose-i-32P on the day of isolation was 7.02 x lo6 cpm per pmole. The glucose-l-3zP was lyophilized and dissolved in 0.25 ml of water.

To produce glucose-l-P labeled with 14C in the glucosyl moiety and 32P in the phosphate moiety, 0.01 mCi of glucose-l-P (glu- case-r4C) was obtained from New England Nuclear Corporation (specific radioactivity = 178 PCi per pmole), evaporated to dryness and dissolved in 0.05 ml of glucose-l-a2P (41.2 pmoles per ml). The specific radioactivity of the compound was determined on the day it was used. Glucose-l-P concentration was determined by enzymatic assay and radioactivity was determined by liquid scintillation counting. Radioactivity originating from 14C and 32P are completely separated at the counter settings used.

iV-Methylhydroxylamine-3H was synthesized according to the method described by Beckmann (14) after exchanging tritium into the starting material. A l.O-ml portion of redistilled nitro- methane (b.p. 100-100.50), 1.0 ml of TsO (1 Ci per g, New Eng- land Nuclear Corporation), and -1 mg of anhydrous sodium acetate was added to a heavy walled test tube containing a magnetic stirring bar. The mixture was frozen and the tube was sealed in an oxygen flame. It was then heated in an oil bath at 80-90” for 24 hours over a magnetic stirrer-heater. The tube was cooled, again frozen, opened, and the contents placed in a 25-ml, round bottomed flask containing a magnetic stirring bar, 0.6 g of NH&l, and 7.0 ml HzO. The flask was fitted with a re- flux condenser and placed in an ice-water bath over a magnetic stirrer. Zinc dust (2.75 g) was added with stirring over a 5-to lo- min period. The mixture was allowed to continue stirring for 2 hours. After 2 hours, the insoluble material was centrifuged and washed twice with HzO. The total supernatant liquid was brought to 100 ml with Hz0 and the pH was adjusted to 5.5 with concentrated HCl. This liquid was then placed on a column (1 x 40 cm) of Dowex 50-X8-Na+ which had been equilibrated with 0.005 M sodium phosphate, pH 5.5. The column was washed until the effluent contained about 3000 cpm per drop. The product was then eluted with 0.1 M NH40H in 0.005 M phosphate. Fractions were brought back to less than pH 6 as soon as they were eluted. The radioactive peak was pooled and adjusted to pH 2 with concentrated HCl. The pool was evaporated to dryness on a rotary evaporator and the product was extracted from the salts with 2-ml aliquots of absolute ethanol until only lo7 cpm were left in the flask. Ether was then added to the ethanol dropwise until the product crystallized. The product was cooled for 30 min in an ice bath and then filtered and air dried. The yield was 360 mg. After recrystallization from ethanol-ether, the final yield was 287 mg. The specific activity was 3.4 x lo6 cpm per pmole and this remained unchanged after recrystallization.

Cochromatography of the tritiated N-methylhydroxylamine. HCl with unlabeled commercial material on Whatman No. 3MM paper with t-butyl alcohol-2 N HCl-water (75:10:15) revealed only one radioactive peak which corn&rated with authentic material. The compound was visualized with silver nitrate stain (10). The melting point of this compound was 83.5-85”. Com- mercial N-methylhydroxylamine . HCl, after recrystallization from ethanol-ether, had a melting point of 86”.

Uniformly labeled sucrose-r4C was obtained from New England Nuclear Corporation and diluted as follows. Uniformly labeled sucrose-14C (0.2 mCi) in ethanol solution at specific activity 370 mCi per mmole was evaporated to dryness on a rotary evaporator and then diluted to 1.1 x 10’ cpm per pmole by the addition of 0.8 ml of 0.04 M sucrose (unlabeled). All uniformly labeled sucrose-14C used was prepared as above unless otherwise stated.

The purity of the commercial uniformly labeled sucroseJ4C was determined by descending chromatography with isopropyl alcohol-water (160:40) as developing solvent. Only one radioactive compound was detected when the paper was assayed for radioactivity with the aid of a 4?r scanner. The sucrose was visualized by staining with silver nitrate (10). The radioactivity migrated with the same RF as sucrose.

Sucrose (fructoseJ4C) was synthesized as described previously (5).

Amino Acid Composition of Sucrose Phosphorylase before and after NaI04 Modification-Amino acid analyses were performed


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1022 Mechanism of Action of Sucrose Phosphorylase Vol. 245, h-o. 5

on samples of sucrose phosphorylase before and after NaI04 modification. Enzyme (1.0 mg) in 0.01 ml of 0.1 M sodium maleate, pH 7, was brought to 0.05 M in NaI04 and incubated at room temperature for 5 min. Under these conditions the enzyme is completely inactivated. The NaIO, was destroyed by the addition of 0.01 ml of ethylene glycol. The inactivated enzyme was dialyzed 24 hours against two 6-liter changes of glass-distilled HzO. The control reaction mixture contained no NaIO.+ This mixture was also dialyzed as indicated for the first sample. Fol- lowing the 24 hour dialysis, the protein solutions were split into two fractions and dried in a vacuum in acid-washed test tubes (13 x 100 mm) over NaOH pellets. To each sample was added 0.25 ml of glass-distilled, constant boiling HCl and the test tubes were sealed under vacuum. The samples were hydrolyzed at 105” in a constant temperature oven for 24 hours and 48 hours. After hydrolysis, the tubes were opened, dried in an evacuated desiccator over NaOH pellets, and submitted for analysis. Anal- yses were carried out on a Beckman amino acid analyzer.

The total cystine-cysteine content of the enzyme before and after NaI04 modification was determined by oxidizing the enzyme with performic acid, hydrolyzing it in acid, and determining the cysteic acid content of the hydrolysate. Enzyme, 0.98 mg, in 0.1 ml of 0.1 M sodium maleate buffer, pH 7.0, was brought to 0.1 M in NaI04 and incubated at room temperature for 5 min. The NaI04 was destroyed by the addition of 0.02 ml of ethylene glycol. The inactivated enzyme was dialyzed 24 hours against two 6-liter changes of glass-distilled water. The control reaction mixture contained no NaI04. This mixture was also dialyzed as indicated for the first sample. Following the 24-hour dialysis, the protein solutions were split into two fractions and dried in a vacuum in acid-washed test tubes (13 x 100 mm) over NaOH pellets. Performic acid was prepared by mixing 0.5 ml of 30% hydrogen peroxide with 9.5 ml of 98 to 100% formic acid and incubating the mixture at 0” for 1 hour. Performic acid (0.2 ml) was added to each sample, the protein dissolved, and the solutions incubated for 2 and 4 hours at 0”. The solutions were then frozen in a Dry Ice-acetone bath and dried in a vacuum over NaOH pellets. Acid hydrolysis and amino acid analysis were performed as described earlier.

Preparation and Isolation of 0-Trimethylsilyl Derivatives of CY- and /I-o-G&ose (15)-A glucoseJ4C sample in 0.1 ml of 0.03 M Pi,

pH 7.0, was diluted with 0.1 ml of mutarotated glucose carrier, (0.03 g per ml) in dimethylformamide. One milliliter of silylating reagent was then added. The silylating reagent contained 4 ml of pyridine (stored over KOH pellets), 1 ml of hexamethyldisi- lazane (Applied Science Laboratories), and 1 ml of trimethylchlo- rosilane (Applied Science Laboratories) mixed just before using. The reaction mixture was incubated at room temperature for 30 min. A precipitate forms, but settles. The supernatant liquid contains the derivatives.

Gas chromatography of 0-trimethylsilyl derivatives was carried out on a column (10 feet X Q inch) of Chromosorb W (AW-DMC S), loo-120 mesh, containing 3% SE-30. The in- strument was the F and M Scientific Corporation model 720, equipped with a thermal conductivity detector. The oven temperature was 180”. The carrier helium flow rate was 120 cc per min. Sample volumes of up to 0.2 ml were injected. The derivatives were collected after chromatography by passing the exit gas through a U-tube containing glass wool immersed in a Dry Ice-acetone bath. The samples were removed from the U-tubes

by washing with 2 ml of pyridine and assayed for radioactivity by liquid scintillation counting.

Chromatographic and Electrophoretic ProceduresElectrophore- sis of glucosyl-peptides was carried out using the procedure and apparatus for high voltage electrophoresis described in detail by Bailey (16). Whatman No. 3MM paper, 55 cm long was used. For pH 3.5 electrophoresis, the origin was placed 20 cm from the positive end of the paper and the samples were subjected to electrophoresis for 50 min at 3 kv (60 volts per cm). The buffer used both for wetting the paper and for electrode-paper contact was pyridine-glacial acetic acid-water (40:400:760), pH 3.5. For pH 1.9 electrophoresis, the origin was placed 12 cm from the positive end of the paper and the samples were subjected to electrophoresis for 35 min at 3.5 kv. The buffer used both for wetting the paper and for electrode-paper contact was 98% formic acid- glacial acetic acid-water (100: 400 :4500), pH 1.9.

Glucosyl-peptides were also separated by ion exchange chromatography. The peptides were placed on a column (0.5 X 45 cm) of Dowex 50-X2 in the pyridinium form which had been equilibrated with 0.3 M pyridine formate, pH 3.2. After glucose and one radioactive peptide are eluted, a convex gradient is applied to the column. The gradient apparatus contained 125 ml of 0.3 M pyridine formate in a constant volume mixing cham- ber which was connected to a reservoir containing 8.5 M pyridine acetate buffer, pH 5.6. This separation procedure has been described in detail for other pepsin peptides (17). Two more radioactive peptides are eluted in the gradient. To separate glucosyl-peptides from glucose, but not from each other, the glucosyl- peptides are placed on the Dowex 50-X2 column described above and equilibrated with 0.05 M pyridinium formate, pH 3.2. After the glucose peak is eluted, 2 M pyridinium formate buffer, pH 3.5, is applied to the column. Under these conditions, all of the radioactive peptides are eluted.

MaterialsSucrose phosphorylase was purified as described previously (5). All other materials were obtained from commercial sources.

RESULTS

Isolation of Glucose-Enzyme Complex by Chemical M edification with Na104-Exposure of sucrose phosphorylase to NaI04 in the presence or absence of substrate leads to loss of enzyme activity. A typical experiment is summarized in Table I. Since it seemed possible that this inactivation was due to the modification of an amino acid, the amino acid composition of the native and NaI04- inactivated enzyme was examined. The only significant difference was a peak found in the inactivated enzyme, which cochro- matographed with cysteic acid. When the inactivated and native enzyme were subjected to performic acid oxidation, a compound was formed from both enzymes, in equal amounts, which, upon amino acid analysis, eluted at the same position as cysteic acid. It was tentatively concluded that NaI04 oxidation converted a cysteine residue to cysteic acid. In subsequent experiments, it was observed that in several enzyme preparations which had been 100% inactivated with NaI04, the amount of cysteic acid found varied from 50 to 100% of that obtained when the native enzyme was oxidized with performic acid. This suggested that cysteic acid formation was not primarily responsible for inactivation. To further test this point, 124 units (4 mg) of enzyme were incubated in 0.4 ml of 0.1 M sodium maleate, pH 7.0, 5 X low4 M NaI04. At 0.5 min, 1.2 min, 2.5 min, and 8 min, 0.1-m] aliquots were removed and added to 0.001 ml of ethylene


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Issue of March 10, 1970 J. G. Voet and R. H. Abeles 1023

glycol to destroy the KaIOe. The tubes were assayed and found to be 79%, 63%, 47%, and 35% active. These samples were subjected to acid hydrolysis and amino acid analysis. In no case was a cysteic acid peak found. Only when the NaI04 concentration was higher did this compound appear. This indicates that the formation of cysteic acid is a phenomenon which is not responsible for the inactivation of sucrose phosphorylase by NaI04.

The inactivation of sucrose phosphorylase by NaIO( suggested that a soluble glucose-enzyme complex might be isolated if NaI04 is added to the enzyme after addition of substrate, i.e. after glucose-enzyme formation. Sucrose phosphorylase was inactivated with NaI04 after addition of uniformly labeled sucrose-14C and the reaction mixture was passed through a Sephadex G-50 column. Radioactive material emerged with the protein peak. A typical experiment showing the elution pattern is shown in Fig. 1. The same results were obtained using a Bio-Gel P-10 column. Results pertaining to the stoichiometry of the reaction are summarized in Table II. Under these conditions, 0.55 to 0.91 pmoles of glucose are bound per lo5 pg of enzyme. When sucrose is added either to the enzyme after NaI04 inactivation or to native enzyme, the amount of glucose associated with the enzyme is approximately lo-fold lower (Experiments 2 and 3, Table II). Experiments with fructose-labeled sucrose and glucose-l-P labeled with 1% in the glucose moiety and with 32P were then carried out to establish whether the whole substrate molecule or only a portion of the substrate is associated with the enzyme (Experi- ments 4 and 5, Table II). These experiments established that only the glucose moiety of the substrate remains associated with the protein. To determine whether the glucose moiety has undergone chemical modification, advantage was taken of the observation, which will be described in more detail below, that the glucosyl-enzyme linkage is unstable at pH 7.0 in 5 M urea and radioactive material is released from the protein, as judged by Sephadex chromatography. NaI04-modified glucose-enzyme complex prepared as described in Fig. 1 containing 27,400 cpm (0.58 mg) was incubat#ed for 16 hours in 0.005 M potassium maleate, pH 7.0, in 5 M urea. After addition of 10 pmoles of glucose carrier, the mixture was passed through a column (1.4 x

100 cm) of Bio-Gel P-2 equilibrated with distilled water. The glucose peak was isolated and its specific radioactivity was 2.5 x 103 cpm per pmole. The glucose was next subjected to descending chromatography with ethyl acetate-pyridine-water (12:5:4) as the developing solvent. The glucose was eluted, and its

TABLE I

Sodium periodate inactivation of sucrose phosphorylase at 25”

Enzyme, 50 units in 0.05 M sodium maleate buffer, was incubated 5 min at room temperature in the presence of the indicated concentrations of either sucrose or NaIO+ or both, in a total volume of 0.2 ml. At the end of the reaction, sufficient ethylene glycol was added to give a final concentration of 1.8 M in order to destroy NaIOa. The mixturewas then assayed for residual enzyme activ-

ity. -

Reaction mixture

Enzyme + 0.01 M sucrose. Enzyme f 0.01 M sucrose, 0.06 M NaI04. _. _. Enzyme + 0.08 M NaIOd. _.

Activity after 5 min

%

92 0.6

<0.06

specific radioactivity determined to be 2.4 x lo3 cpm per pmole. The specific activity therefore remains constant through two purification procedures. The total radioactivity found in the glucose was 24,500 cpm which accounted for 90% of the radio-

5 IO 15

Tube No.

FIG. 1. The isolation of a NaIOa-modified glucose-enzyme complex on Sephadex G-50. A solution containing 250 units per ml of enzyme in 0.01 M sodium maleate buffer, pH 7.0, 0.01 M uniformly labeled sucrose-i4C of specific activity 1.1 X lo7 cpm per pmole, and 0.05 M NaI04 in a final volume of 0.2 ml was incubated for 5 min at room temperature. The solution was then made 1.8 M in ethylene glycol to remove excess NaI04, assayed for residual enzyme activity, and placed on a column (1 X 50 cm) of Sephadex G-50 equilibrated with 0.005 M potassium maleate buffer, pH 7.0. After the void volume had passed through the column, l-ml fractions were collected.

TABLE II

Amount of glucose bound to sucrose phosphorylase after RaIOd

modi$cation in presence of sucrose

Enzyme, 50 units, in 0.05 M sodium maleate, pH 7, was incu-

bated for 5 min at room temperature in the presence of 0.01 M of the indicated substrate and 0.06 M NaI04. The final volume was 0.2 ml. Substrates were added in the order in which they are

listed in the table. At the end of the reaction, the NaI04 was destroyed by the addition of enough ethylene glycol to make the final concentration 1.8 M. The mixture was assayed for residual activity. The amount of protein-bound radioactive material

was determined as in Fig. 1. Specific activity of substrate: uniformly labeled sucrose, 3.3 X lo7 cpm per pmole; fructose- labeled sucrose, 9.4 X 106 cpm per Mmole; glucose-14C-1-32P, SzP,

5.5 X lo6 cpm per pmole; W, 1.4 X 10’ cpm per pmole.

Reaction mixture Micromoles of glucose/1oo,000 fig of enzyme

1. Enzyme, uniformly labeled sucrose- l*C, NaI04..

2. Enzyme, NaI04, uniformly labeled sucrose-i% (sucrose added after inactivation). . .

3. Enzyme, uniformly labeled sucrose- 14c................................

4. Enzyme, sucrose-W (fructose-labeled) . . . .

5. Enzyme, glucose-W-1-3zP, NaI04.

%

0.6

0.6 0.067

0.067

0.015 W 0.64 3ZP 0.014

0.55-0.91


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1024 Mechanism of Action of Sucrose Phosphorylase Vol. 245, No. 5

activity originally present in the modified glucose-enzyme complex. This result indicates that the glucosyl moiety of the complex remains intact during modification by NaI04.

Reactions of NaIOA-Modijied Glucose-Enzyme Complex--Ex- periments were carried out to determine whether the complex shows any enzyme activity, i.e. whether it can transfer the glucose moiety to an acceptor. Incubations were carried out in the presence of phosphate, fructose, and xylose, and without added acceptor, so that the reaction with water could occur. Xylose is not a glucosyl acceptor in the transfer reaction catalyzed by sucrose phosphorylase. The results are summarized in Table III. The complex, or at least a fraction of the molecules of the complex, can transfer glucose to an acceptor. The NaIOd-treated enzyme still shows substrate specificity, qualitatively similar to that of the native enzyme, but the reaction proceeds at a greatly reduced rate. At the present time we have no explanation of why only a fraction of the glucose associated with the enzyme is subject to release either through transfer to an acceptor or hydrolysis. Possible reasons will be considered later.

The observation that the modified enzyme shows substrate selectivity suggests that the tertiary structure of the protein plays a role in the reactions of modified glucose-enzyme complex. To test this point, the following experiment was carried out. NaIOd-modified glucose-enzyme complex was prepared and isolated as described in Fig. 1, except that the specific radioactivity of the uniformly labeled sucrose-l% was 4.4 x 10’ cpm per pmole. To a test tube containing 0.42 g of urea (7 mmoles) and 0.1 ml of 1.0 M potassium phosphate buffer, pH 7.0, was added 0.6 ml of a solution containing the modified glucose-enzyme complex (2.6 x 104 cpm). The reaction mixture was brought to a final volume of 1.0 ml. A parallel reaction was carried out which contained no urea. The mixtures were incubated for 24 hours, and 8.9 pmoles of unlabeled glucose-l-P carrier were added to each. The solution was subjected to chromatography on a column (1 x 15 cm) of Bio-Gel P-10 equilibrated with 0.005 M potassium maleate, pH 7. The low molecular weight fraction was pooled and the

resulting solution was applied to a 1.5-ml Dowex l-Cl- column equilibrated with water. The column was washed with 10 ml of 0.01 M glucose and 5 ml of water. The glucose-l-P was then eluted with 0.1 M LiCl. In the absence of urea, the glucose-l-P isolated contained 6100 cpm. In the presence of urea there were only a total of 350 cpm associated with the glucose-l-P. There- fore, destruction of the teritary structure of the NaIOI-modified glucose-enzyme complex by urea destroys its ability to react with Pi to form glucose-l-P.

The stability of the glucose-enzyme complex was determined under a variety of conditions in urea and buffer by reisolating the protein at various times and redetermining its specific radioactivity (Table IV). The ability to form glucose-l-P as a function of time was also measured (Table V) . In urea, the complex is relatively stable at pH 4.7, but is essentially completely decomposed at pH 10.5 by the time the first point (15 min) is taken. At pH 7, in the absence of urea, as observed before, not all of the glucose associated with the protein is released. In the presence of urea, all of the glucose can be released. Semilog plots of the rates of decomposition of the complex at pH 7 in buffer and in urea are shown in Fig. 2. The data again suggest the presence of at least two kinds of molecules with different reactivities.

Isolation of Glucose-Enzyme Complex by Precipitation with 6 M Ammonium Formate, pH S.&The acid stability of the NaI04- modified glucose-enzyme complex suggested that a glucose-enzyme complex might be isolated under acidic conditions without prior NaIO., treatment. Table VI shows the results of experiments in which the enzyme was incubated with uniformly labeled sucroseJ4C and the protein was precipitated by the addition of 5 M ammonium formate, pH 3.0. The precipitated protein, after extensive washing, was radioactive. When D-fructose-labeled sucroseJ% was used instead of uniformly labeled sucrose-l%, the precipitated protein had only negligible radioactivity. This result indicates that only the n-glucose moiety of sucrose becomes firmly bound to the enzyme. From the radioactivity associated with the protein in several experiments it can be calculated that

TABLE III

Reactions of NaIOd-modijed glucose-enzyme complex at pH Y.0

The modified glucose-enzyme complex was prepared and iso- In Reaction a, glucose was purified by Solvent 1 followed by lated as in Fig. 1. Immediately upon isolation, the modified Solvent 2. In Reaction b, glucose and glucose-l-P were sepa-

glucose-enzyme complex was mixed with 0.005 M potassium male- rated on Solvent 3. Glucose was further purified on Solvent 2 ate, pH 7.0, containing: Reaction a, no addition; Reaction b, and glucose-l-P by paper electrophoresis. In Reaction c, glucose

0.01 M Pi, pH 7.0; Reaction c, 0.01 M fructose; Reaction d, 0.01 M and sucrose were separated on Solvent 1. Both products were

xylose. These reaction mixtures were incubated for 16 hours. then purified on Solvent 2. Reaction mixture d was subjected to The reaction mixtures were streaked on Whatman No. 3MM Solvent 1. Glucosyl-xyloside would have separated from glucose

paper. The following solvent systemswere used: Solvent 1, ethyl in this solvent system. Only glucosewas observed. Glucosewas

acetate-pyridine-water (12:5:4); Solvent 2, isopropanol-water further purified on Solvent 2.

(160:40) ; Solvent 3, acetone-30% acetic acid (1: 1). Paper electro- The specific radioactivities of the products remained constant ihoresis- was carried out for 3 hours at 400 volts using 0.5 through successive purifications.

M NH40H as buffer.

Reaction mixture Total

radioactivity in complex

a. Glucose-enzyme + Hz0 b. Glucose-enzyme + Pi

em 23,000 14,300

c. Glucose-enzyme + fructose 14,700

d. Glucose-enzyme + xylose 14,700

Products recovered Radioactivity in product

Glucose

Glucose Glucose-l-P Glucose Sucrose

Glucose Glucosyl-xylose

cm 10,700

3,700

2,200 8,400 2,140

9,400 Not detected

Complex converted to

product

% 47

41

72

64


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TABLE IV Specijic radioactivity of NaIOr-modified glucose-enzyme complex as

function of time under various conditions A 0.15-mg portion of NaIOa-modified glucose-enzyme complex

(specific activity 0.82rmoles of glucose per 100,OOO~g of protein) was incubated in 1.0 ml of 7 M urea in the appropriate buffer. At the specified time the mixture was passed through a column (1 X 15 cm) of Bio-Gel P-10 equilibratedwith 0.005 M K+-maleate, pH 7.0, to remove small molecules and reisolate the protein. The protein remained on the column 6 min after the specified time under these conditions. The buffers used in the incubation mixtures were 0.1 M potassium acetate, pH 4.7; 0.005 M potassium maleate, pH 7; 1 M potassium bicarbonate, pH 10.5.

Time

hrs

0

0.25

0.5 0.7 1.0 1.5 4.0

24.0 48.0

24 hrs in buffer + 24 hrs in urea

-

_ _

:.5 x 10’ 3.5

3.6

3.3

2.6

14

-

:.3 x lo’

2.3

1.3

0.54

0.31

TABLE V where a = 48 hours for buffer and 24 hours for urea.

.3 x 10

3.6

3.3

2.7

2.3 1.9 0.65

-2

4.5 t I I I ‘p

0.15 I 2 3 4

0.13 Time (Hrs.)

FIG. 2. Semilog plot of the percent of NaIOb-modified glucose- 0.14 enzyme complex remaining as a function of time. - - -, 0.005 M

potassium maleate, pH 7.0; -, 7 M urea containing 0.005 M po-

0.12 tassium maleate, pH 7.3. X represents points determined as in Table V and 0 represents points as determined in Table IV. Percentage glucose-enzyme complex remaining was taken as

(cpm per mdt - (cpm per md, (cpm per mg)0 - fcpm per mg),

Stability of NaIOh-modified glucose-enzyme complex as measured by

ability to form glucose-l-P

NaIOd-modified glucose-enzyme complex (1.4 ml) containing 14,300 cpm was incubated in 0.005 M potassium maleate buffer at pH 7.0. At the specified time, 0.2 ml of 0.1 M Pi at pH 7.0was added and the mixture was incubated for 7 hours. Glucose-l-P (9.35 &moles) was then added and the reaction mixture was purified chromatographically and electrophoretically as described in Table III. The amount of glucose-l-P found when Pi was added at zero time is designated as 100.

Zero time.. 20 min. 40min.......... 1 hour. . . 2 hours. 4 hours.

.........

.........

.........

.........

Time of addition of Pi I

Glucose-l-P recovered

100 . 79

64 . 48

38 32

0.67 to 1.2 pmoles of glucose are incorporated per 100,000 pg of protein on acid precipitation.

To examine the nature of the interaction between the radioactive moiety and the enzyme in the acid-precipitated glucose- enzyme complex, experiments were conducted to determine whether the radioactivity remained associated with the protein after solubilization of the complex in urea. Enzyme (40 units) in 0.3 ml were incubated with uniformly labeled sucrose-W and the protein was precipitated and washed under the conditions described in Table VI. The resulting precipitate, which had a specific activity of 3.9 x lo4 cpm per mg, was dissolved in 0.7 ml of 0.03 M ammonium formate, pH 3, containing 5.7 M urea. The solution was incubated for 10 min at room temperature. In

Amount of glucose bound to sucrose phosphorylase after acid

TABLE VI

precipitation in presence of sucrose The reaction mixture contained 1 mg of enzyme, 3 X 10-S M

potassium maleate buffer, pH 7.0, and 1.3 X 10-z M sucrose-%. The final volume was 0.15 ml. The reaction was carried out at 25". After 5 set, 2.0 ml of 5 M ammonium formate, pH 3.0, was added to the reaction mixture. The precipitated enzyme was washed seven times with 5-ml aliquots of 0.25% formic acid. The specific radioactivity of the precipitate was determined by dissolving it in 0.1 ml of 1.0 M NaOH and using diquots of this solution for the determination of protein by the method of Lowry et al. (13) and determination of radioactivity by scintillation counting.

Additions to reactions Radioactivity GlUCOSf2

Uniformly labeled sucrose-14C (1.2 X 10’ cpm/fimole) . . . . . . . . . 4.0-7.8 0.67-1.2

Sucrose-W labeled in the fructose moiety (9.4 X lo6 cpm/pmole) . . . . . 0.6 0.051

order to separate any small molecules from the proteinit was then chromatographed on a column (1 X 15 cm) of Bio-Gel P-10 equilibrated with 0.005 M K+-maleate, pH 7.0. The protein fractions were pooled and the specific activity of the protein was determined to be 3.5 x lo4 cpm per mg. Of the radioactivity which is precipitated with the enzyme on the addition of acid, 90% remains associated with the protein even when it is dissolved in urea. This result suggests that the radioactive moiety is covalently bound to the protein.


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1026 Mechanism of Action of Sucrose Phosphorylase Vol. 245, Ko. 5

IO 0 I I

IO cm or,p,n +

FIG. 3. Electrophoresis of pepsin digest of NaIOt-modified glucose-enzyme complex at pH 3.5 for 50 min at 3 kv.

100 IA

90 r

cn L II II

10 - Gl”COM 0 IO cm Owl” +

FIG. 4. Electrophoretic pattern at pH 3.5 of pepsin digest of acid-precipitated glucose-enzyme complex. A, glucose-enzyme complex, 4.7 mg, incubated for 1.5 hours with 1.5 mg of pepsin in 0.9 ml of 0.16% formic acid, pH 3.0; B, glucose-enzyme complex, 1 mg, incubated for 1.5 hours with 1 mg of pepsin in 0.7 ml of 0.03 M ammonium formate, pH 3.0; C, glucose-enzyme complex, 1 mg, incubated for 4 hours with 2 to 4 mg of pepsin in 0.3 ml of 0.25% formic acid, pH 3.0; D, 0.05 ml of digest (A) brought to pH >lO by the addition of 0.001 ml of concentrated ammonium hydroxide, incubated for 5 min, and brought back to pH 3.0 by the addition of 0.001 ml of 9Oclc formic acid.

Formation of Glucosyl-Peptides by Pepsin Digestion of Glucose- Enzyme ComplexesThe acid stability of the glucose-enzyme linkage suggested that pepsin digestion of either the NaIOI-modified glucose-enzyme complex or the acid-precipitated glucose-

enzyme complex might result in the formation of glucosyl-peptides which could be isolated and studied. NaIOI-modified glucose-enzyme complex (2.5 ml) containing 0.63 mg of protein of specific radioactivity 4.5 x lo4 cpm per mg was mixed with 0.4 ml of 1 M potassium acetate buffer, 1’11 4.0. The protein precipitated. This precipitate was centrifuged and suspended in 1 ml of 0.25% formic acid, $1 3.0. Pepsin, 0.1 mg, was added and the suspension was allowed to incubate at room temperature for 48 hours. The remaining precipitate was removed by centrifugation and the supernatant fluid was evaporated in a vacuum over NaOH pellets until the volume was reduced to 0.5 ml. The sample was then subjected to paper electrophoresis at pH 3.5 for 50 min at 3 kv. Fig. 3 is a radioactivity scan of the pH 3.5 elec- trophoretogram. Four positively charged entities can be clearly distinguished from the neutral peak near the origin. The peak near the origin, which migrated the same distance as glucose, was assumed to be free glucose, but was not further identified. There is no positive proof that these entities are peptides. However, the facts that more than one peak is produced on digestion with a nonspecific protease and that these peaks are positively charged but of different mobilities at ~113.5 are good indications that they are peptides. The glucosyl-peptides may be overlapping or the glucose may have migrated to several sites on the protein.

The acid-precipitated glucosyl-enzyme complex was also subjected to pepsin digestion. Enzyme, 250 units (5 mg), in 0.6 ml, was incubated with 0.3 ml of 0.04 M uniformly labeled sucroseJ4C and the protein was precipitated and washed as described in Table VI. The resulting glucose-enzyme complex was lyophilized and dissolved in 0.4 ml of performic acid (0.95 ml of 97% formic acid plus 0.05 ml of 30% hydrogen peroxide incubated for 1 hour at 4”). The solution contained 258,000 cpm or 0.047 pmoles of glucose-enzyme complex. The complex was incubated for 1 hour at 0”, evaporated to dryness in an evacuated desiccator containing NaOH pellets, and suspended in 0.9 ml of 0.16% formic acid containing 1.5 mg of pepsin. The suspension was incubated at 37” for 1.5 hours with frequent stirring, Any remaining precipitate was removed by centrifugation in a clinical centrifuge at top speed for 5 min. The resulting pepsin digest contained 132,000 cpm or 0.024 pmoles of glucosyl units. This result cor- responds to the recovery of 5070 of the radioactivity originally associated with the complex. In similar pepsin digestions up to 90% of the radioactivity was recovered in soluble form.

An aliquot of the pepsin digest was subjected to paper electrophoresis at pH 3.5 for 50 min at 3 kv. Fig. 4R is a radioactivity scan of the paper on which this electrophoresis was performed. Four positively charged peptides can be clearly distinguished from the neutral peak near the origin.

This glucosyl-peptide pattern is identical with the pattern obtained on pepsin digestion of the NaI04-modified glucose-enzyme complex, although the relative amount of each peptide varied. Peptide 3, as well as Peptide 4, can be eliminated by altering the length of pepsin hydrolysis and the amount of pepsin added (Fig. 4, B and C). These changes in peptide pattern are presumably due to changes in the extent of pepsin digestion. The fact that electrophoretically similar peptides can be obtained from the pepsin digestion of acid-precipitated glucose-enzyme complex and NaI04-modified glucose-enzyme complex suggests that the glucose is associated with the same residue in both complexes.

Since exposure to alkali caused the rapid release of glucose from the NaIOd-modified enzyme, the base lability of the glucosyl-peptides was examined. The peptides were incubated in 0.3 M


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NHaOH for 5 min, reacidified with 900/O formic acid to pH 3.0, and then subjected to paper electrophoresis at pH 3.5. The results are shown in Fig. 40. Only a neutral peak is seen, suggest- mg that glucose was released under these conditions. Similar results were obtained with peptides derived from the NaI04- treated complex. The similar base lability of the glucosyl-peptides derived from acid-precipitated and NaIOrmodified glucosyl-enzyme suggests that the same type of bond is present in both complexes.

Identification o,f Radioactive %lolecule Associated with Peptides Derived from Acid-Precipitated Glucose-Enzyme Complex--Radio- active peptides, 0.06 ml, derived from acid-precipitated glucose- enzyme complex of specific activity 4.6 x lo4 cpm per mg and containing 10,800 cpm, were lyophilized together with 10.3 pmoles of unlabeled glucose and dissolved in 0.4 ml of 0.1 M pyridine formate, pH 6.2. The mixture was incubated for 24 hours at room temperature. Under these conditions, the radioactive substance is released from the peptides. The mixture was evaporated on a rotary evaporator, taken up in a small amount of water, and applied to Whatman No. 3MM paper in a 1.5-inch streak. Descending chromatography was carried out for 16 hours with n-butyl alcohol-acetic acid-water (12:3:5) as the developing solvent. The radioactive glucose peak was eluted and its specific radioactivity was found to be 998 cpm per pmole. If all of the radioactivity associated with the peptides was due to glucose, the specific activity should be 1050 cpm per pmole. The eluate was concentrated on a rotary evaporator, applied to What- man No. 3MM paper, and subjected to descending paper chromatography using isopropyl alcohol-water (160:40) as the developing solvent. After this chromatographic purification, the specific radioactivity of the glucose was 1250 cpm per pmole. The glucose sample was evaporated to dryness on a rotary evaporator and converted to glucose-6-P by incubating for 2 hours at room temperature in the presence of 0.22 ml of 0.0013 M Tris-chloride, pH 8.0, containing 0.01 M neutralized ATP, 6 X lo-* M MgS04, and 0.01 mg of hexokinase. The mixture was then applied to Whatman No. 3MM paper in a l&inch streak and subjected to ascending chromatography for 6 hours using methanol-ammonia- water (6: 1:3) as the developing solvent. Under these conditions, glucose and glucose-6-P are separated. The specific radioactivity of the glucose-6-P was found to be 1090 cpm per pmole. These results establish that the material released from the radioactive peptides was n-glucose.

Reactivity of Glucosyl-Peptides-In order to gain more informa- tion concerning the nature of the glucose-pept#ide linkage, experiments were conducted to explore the stability of this bond. Table VII contains the results of a survey of the stability of the glucosyl peptides as a function of pH. It can be seen that at acid concentrations up to 1 M HCl the glucosyl-peptides are relatively stable. As the pH is increased the glucosyl-peptide linkage becomes increasingly unstable. At pH 8.0, it is completely decomposed in 10 min. A more careful study was made of the rate of decomposition of the glucosyl-peptides in the intermediate pH range. The effect of N-methylhydroxylamine upon their rate of decomposition was also examined, since it was hoped this reagent might be used to label the binding site. The decomposition of glucosyl-peptides proceeds more rapidly in N-methylhydroxylamine at both pH 5.0 and 6.0. At pH 5.0, the t+ for the reaction was 360 min in pyridine and 120 min in N-methylhydroxylamine. At pH 6.0, the t+ for the reaction was 80 min in pyridine (1 M)

and 10 min in N-methylhydroxylamine (1 M). The first order

TABLE VII

Stability of glucosyl peptides as function of pH

Acid-precipitated glucose-enzyme complex, 313 mg, of specific radioactivity 2.5 X 104 cpm per mg, was oxidized with performic acid for 1 hour, digested with 3 mg of pepsin in 0.6 ml of 0.25”j;

formic acid for 4 hours, and lyophilized in aliquots containing 3000 cpm. Each aliquot was incubated for a given amount of time in 0.05 ml of buffer at the appropriate pH and then diluted to 2.0 ml with 0.05 M pyridine formate, pH 3.4. Unlabeled

glucose (10 pmoles) was added and the solution was mixed with 1.0 g of filter-dried Dowex 50-X2 in the pyridinium form. Glu- cosyl-peptides are quantitatively absorbed on this resin. The

mixture was filtered and the specific radioactivity of the glucose in the filtrate determined by Glucostat and scintillation counting. The percentage glucosyl-peptides remaining was taken as

100 - ( c p m in glucose), - (cpm in glucose)0

(cpm)totai - (cpm in glucose)0 x 100

The buffers used were pII 1.9, 98% formic acid-glacial acetic acid-water (4:40:760); pH 6.0, 0.01 M Tris-maleate; pH 8.0, 0.01

M Tris-maleate.

Time

Glucosyl-peptides remaining

1~Hcl 1 pH 1.9 1 pH 3.5 1 pH 6.0 1 pH 8.0

10 min..

2.5 hr 87

18.5 hr.......

rate plots do not extrapolate to 100% complex. The extrapola- tion point varies and would indicate that there may be a glucosyl- peptide which decomposes more rapidly than the others under the experimental conditions and that the percentage of glucose in this reactive peptide varies from one peptide preparation to another. The existence of a variation in the distribution of glucosyl-peptides is also suggested by the variability of the relative peak heights in electrophoretic patterns.

The glucosyl-peptide bond becomes unstable at pH values above 5 and decomposes rapidly at pH 8.0. This instability above pH 5.0 is quite similar to that observed with the glucosyl enzyme.

Site of Cleavage of Glucosyl-Peptide Bond-In the over-all reaction catalyzed by sucrose phosphorylase, bond cleavage occurs between C-l of the glucose moiety of the substrate and the glycosidic oxygen. Therefore, the glucose-enzyme complex no longer contains the original glycosidic oxygen. When glucose is released from the glucosyl-enzyme or from derived peptides, the C-l hydroxyl group of glucose is therefore contributed either from the enzyme or the solvent. The solvolysis of the glucosyl- peptides was carried out in 75% methanol to distinguish between these two possibilities. If the product of this reaction is methyl glucoside, then the oxygen of glucose is derived from the solvent. I f no methyl glucoside is formed, then the oxygen is derived from the enzyme. These conclusions are based on the assumption that the chemical properties of water and methanol are very similar in solvolysis or replacement reactions. The assumption is justified in view of the similarity in acid strength and nucleo- philicity. The results are summarized in Table VIII. Essen- tially no methyl glucoside was formed; therefore, the oxygen of

the glucose released from glucosyl-peptides is derived from the enzyme, and the glucose must be linked to an oxygen-containing


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TABLE VIII

Products of methanolysis of glucosyl-peptides at pH 6 Glucosyl-peptide, 7.5 X 10e3 pmoles, 10 pmoles of carrier

glucose, and 10 pmoles of carrier a-methylglucoside were ,incubated for 2.5 hours in 0.03 ml of a solution of 75% methanol containing 1.5 M pyridinium formate, pH 5.8. The solution was

then diluted to 10 ml with 0.05 M pyridine formate, pH 3.4. Dowex 50-X2 (pyridinium form) (5 g) was added and the mixture was filtered. The filtrate was evaporated to dryness in a des-

iccator. Glucose and or-methylglucoside were separated from each other by paper chromatography in isopropanol-water (160:40) and purified by paper chromatography in n-butyl alco-

hol-acetic acid-water (120:30:50) and ethyl acetate-pyridine- water (120:50:40).

The specific radioactivity of glucose remained constant through the two purification procedures. The specific radioactivity of

the or-methylglucoside decreased after each purification. The radioactivity reported is the maximum amount that could be present in this compound.

Total radioactivity in peptides (cpm) 34.4 x 103 Radioactivity released (%). . 60 Radioactivity in glucose (cpm). 23.8 x 103 Radioactivity in a-methylglucoside (cpm) . . <82

functional group of the enzyme. Furthermore, hydrolysis of the glucosyl-peptides does not involve solvent attack at C-l of the peptide-bound glucose so that the configuration at C-l of the glucose released must be the same as that of glucose bound to the peptides. This fact enabled us to establish the configuration of the glucose-peptide linkage. Glucosyl-peptides were incubated under conditions where glucose was rapidly released. The glucose was then converted to the 0-trimethylsilyl derivative. The derivatized cy and /3 anomers of glucose could be separated by gas chromatography. The separations achieved for authentic a,&n-glucose are illustrated in Fig. 5. These data also show that no significant anomerization occurs during the derivative formation. The results from a typical experiment are shown in Table IX. Essentially all of the radioactivity released was found with the /?-glucose derivative indicating that glucose released had the @ configuration. Any cY-glucose present could be accounted for by anomerization of the released ,&glucose prior to derivative formation. A 3-hour point was included to test the validity of the method. After 3 hours complete mutarotation occurs and the reaction mixture should, and does, contain 61 y0 p isomer and 39 ‘% a isomer. These experiments show that essentially all of the glucose released from the peptides has the fl configuration. This result, together with the results of the solvolysis experiments, establish that glucose is linked to the protein through a ,8 linkage and formation of the glucosyl-enzyme proceeds with inversion of configuration at the C-l carbon of the substrate.

Abortive Attempts to Identify Chemical Linkage between Glucose and Enzyme-A number of experiments were conducted which were designed to label the functional group to which the peptide is linked. These experiments were unsuccessful and will not be described in detail. The first experiments involved reaction of glucosyl-peptides with N-methylhydroxylamine. The acceleration of the rate of glucose release in the presence of N-methylhydroxylamine suggested that this reagent might be used to label the point of attachment of glucose to the protein. Glucosyl- peptides were therefore treated with iV-methylhydroxylamine-3H

‘- A 9

h 8

Gas-Liquid Chromatography of

Penta-0-TMSi-#-D-Glucose Pyranose

FIG. 5. Gas chromatography of 0-trimethylsilyl derivatives. A, &n-glucose, 3 mg, was dissolved in 0.1 ml dimethylformamide. A 10-J portionof 0.03 M potassium phosphate buffer, pH 7, was then added followed immediately by 1 ml of trisilylating reagent; B, p-n-glucose, 3 mg, was dissolved in 10 pl of 0.03 M potassium phosphate buffer, pH 7, and allowed to mutarotate overnight. Di- methylformamide (0.1 ml) was added followed by 1 ml of trisilylating reagent.

and reisolated by Dowex 50 chromatography. Two control experiments were carried out in which, in one case, peptides were those prepared from an enzyme which had not been exposed to sucrose and, in another case, peptides were those from which the glucose had been removed by prior base treatment. The amount of N-methylhydroxylamine-3H associated with the reisolated peptides was the same in all cases. We therefore assumed that the amount of radioactivity found with the peptides represented nonspecific labeling. Possible reasons for the failure to observe specific incorporation are (a) N-methylhydroxylamine does not replace glucose and the rate acceleration observed for the release of glucose was due to general base catalysis, and (b) an adduct between the peptides might have been formed but was itself unstable under the conditions of the reaction.

An attempt was made to treat the glucosyl-peptides with methanolzH in order to see whether methanol was incorporated on methanolysis. It was found that the amount of radioactivity associated with the peptides far exceeded the amount which could be incorporated if each mole of glucose were replaced by 1 mole of methanol. The results of this experiment were, therefore, inconclusive. The radioactivity could have been due to either nonspecific reaction of the peptides with methanol-aH or the presence of reactive radioactive impurities.

Since glucose might be linked to the enzyme through an ester linkage, an attempt was made to reduce this bond with lithium


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TABLE IX Amount of ol-o-glucose and p-o-glucose formed on hydrolysis of

glucosyl-peptides

Glucosyl-enzyme was prepared by acid precipitation as described. The precipitated enzyme contained 0.72 pmoles of glucose per 100,000 rg of enzyme and the specific radioactivity was 2.7 X lo8 cpm per pmole. Glucosyl-peptides were prepared from the glucose-enzyme complex by pepsin digestion. Lyophi- lized aliquots of glucosyl-peptides containing 52,000 cpm were allowed to decompose in 10 ~1 of 0.03 M Pi, pH 7.0 or 7.7. At time t, 1-J aliquots were diluted to 2 ml with 0.05 M pyridinium formate, pH 3.2, and the percentage glucose releasedwas determined as in Table VII. Mutarotated glucose carrier in dimethylformamide (0.1 ml, 0.03 g per ml) was then added to the remaining reaction mixture and the 0-trimethylsilyl derivatives were prepared immediately. For the zero time point, 0.1 ml of glucose carrier was added to the lyophilized aliquot, the tube was main- tained at O”, and 10 ~1 of 0.03 M phosphate was then added. A lo-p1 aliquot was diluted to 2 ml with 0.05 M pyridinium formate, pH 3.2, to determine the percentage glucose present. The re- mainder was trisilylated. The glucose derivative (CY and p iso- mers) was then isolated by gas chromatography. The fraction of glucose present as OL isomer was calculated as follows

total radioactivitv in a! Desk Ly lSomer = total radioactivity (o( + p)

From this amount and the amount of a-glucose present at zero time, the fraction of or-glucose present in the released glucose was determined.

To determine the amount of ol-o-glucose formed from authentic p-n-glucose under these reaction conditions, p-n-glucose, 3 mg, was dissolved in 10 ~1 of 0.03 M Pi, pH 7 or 7.7. At time 1, 0.1 ml of dimethylformamide was added and the mixture was treated as above. The percentage O-trimethylsilyl-a-n-glucose present was determined by measuring the area under the curves obtained from the gas chromatograph.

Percentage a = area,

area, + areag

Tie of hydrolysis PH

Total reactivity

%E.i glucose

I I

Released glucose present

as LI isomer

a-m

B 1lWXe armed from

authentic p-n-

glucose

% Zero time. ........ 7.0 21 5min ........... 7.0 69 97 3 9 5rnin ............ 7.6 82 92 8 15 3 hrs ............. 7.0 100 61 39 39

borohydride-aH (18). Again, the amount of tritium incorporated far exceeded the expected amount. In a control experiment, peptides, from which glucose had been removed, were treated with LiBaH* under identical conditions. Approximately the same amount of radioactivity was incorporated into these peptides as into the glucose-containing peptides. After LiBaH., treatment, both sets of peptides were hydrolyzed and the distribution of radioactivity among the resulting amino acids was examined. No significant difference could be detected and the approach was not pursued further.

DISCUSSION

Two basic properties of the double replacement mechanism postulated for sucrose phosphorylase are: (a) the cleavage of the

glycosidic bond prior to reaction with acceptor; (b) the formation of covalent glucosyl-enzyme. Experiments described here support both of these aspects of the mechanism. The isolation of a complex containing only the glucosyl moiety of substrate is evidence for the cleavage of the glycosidic bond in the absence of acceptor. Absence of the aglycon from the complex was demon- strated by experiments in which the aglycon portion of the substrate was labeled with 14C or 32P. Several lines of evidence establish the presence of a covalent bond. (a) The complex obtained after NaI04 inactivation as well as the complex obtained by acid treatment could be passed through a Sephadex column without loss of the glucosyl moiety. Exposure to 6 M urea in acid did not cause release of the glucose. (b) The transfer of the glucosyl moiety from the NaIOd-inactivated complex to acceptors such as phosphate or fructose indicates that an acti- vated form of glucose must be present. Glucose associated non- covalently with protein could not react to form glucosides. (c) Treatment of both types of glucosyl complexes with pepsin re- sulted in the formation of glucosyl-peptides. (d) All of the glucose released from glucosyl-peptides was of the /3 configuration. The absence of mutarotation indicates that free glucose was not present.

The double replacement mechanism also predicts the stereochemistry of the intermediate glucose-enzyme complex. This complex is formed as a result of replacement of the aglycon of the substrate by a nucleophilic group of the enzyme. It is highly likely that this reaction involves an inversion of configuration at C-l of glucose, so that the glucose becomes linked to the protein through a /3 linkage. Experiments described here establish the presence of a /3 linkage in glucosyl-peptides obtained from the glucosyl enzyme and therefore confirm the stereochemical pre- dictions of the double replacement mechanism. All experimental evidence obtained so far is consistent with the double replacement mechanism; no contradictory evidence has been obtained.

The glucosyl-enzyme complex obtained upon NaI04 treatment of sucrose phosphorylase is of special interest since it possesses transfer activity and acceptor specificity similar to that of

the native enzyme, but reacts at approximately Z&0 the rate of the native complex. The ability of this complex to transfer the glucosyl moiety is dependent upon tertiary structure, since the transfer reaction no longer occurs in 6 M urea. The mechanism of action of NaI04 is unknown. Possibly NaI04 modifies an amino acid component of the protein so that structural changes in or about the active site occur. Alternatively, one of the general acid or base components of the active site is modified so that it can no longer effectively participate in the catalytic process.

It was noted that the NaI04-modified enzyme in the absence of urea did not release all of its glucose either by transfer or by reaction with water, whereas in the presence of urea, where all transferase activity is lost, all of the glucose can be released by hydrolysis. We interpret these results to indicate that two species of NaI04-modified enzyme are present. One still contains catalytically active enzyme with greatly reduced catalytic efficiency. This enzyme will release glucose by either transfer to water or to some other acceptors. The remaining enzyme has been more extensively modified, or modified in different ways, so that it can no longer transfer glucose to acceptors. The glucosyl unit is unreactive and can only be released when the tertiary structure of the enzyme is destroyed. We believe that the release of glucose in the presence of urea is a nonenzymatic proc-


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ess and merely represents the susceptibility of the glucosyl- enzyme complex to base hydrolysis.

To obtain a more detailed understanding of the mechanism of action of sucrose phosphorylase, the nature of the linkage between glucose and the protein must be identified. Whenever an enzyme-substrate complex is isolated and it cannot be demon- strated that such a complex meets the kinetic requirements of a functional intermediate in the over-all catalytic process, the question must naturally be raised whether the substrate is bonded to the same linkage as in the catalytically active complex. It is conceivable that during modification of the complex, the glucosyl group has migrated. This possibility, of course, cannot be eliminated. There are several observations which indicate that migration of the glucosyl group may not have taken place. The peptides isolated from glucosyl enzymes prepared two en- tirely different ways appear to be very similar and to have very similar chemical properties. It is unlikely that under two different sets of conditions, identical migrations would have occurred. Furthermore, the stereochemistry of the intermediate is as expected from a double replacement mechanism. In addition, the NaIO&-modified complex possesses transferase activity and acceptor specificity similar to the unmodified enzyme. We consider the probability that glucose is attached to a catalytically unimportant functional group sufficiently unlikely to warrant further exploration of the nature of the glucose-enzyme linkage.

In considering the possible type of linkage between glucose and the protein, the following facts must be taken into consideration. (a) The C-l atom of glucose is bonded through an oxygen atom to the protein since solvolysis of glucosyl-peptides in methanol did not form methyl glucoside. (b) The glucosyl-protein bond as well as the glucosyl-peptide bond is extremely base labile. At pH 6, t+ for hydrolysis of the glucose-peptide bond is about 80 min. At pH 8, the bond is completely decomposed in 10 min. These facts limit to three types the possible types of bonds which can occur. Glucose could be linked to the protein through the hydroxyl group of serine or threonine. Another possibility is that the glucose is linked through an imidate linkage which could be formed by interaction of one of the amide carbonyl groups of the protein with a carbonium ion at C-l of glucose. Finally, glucose could be bonded to the protein through an ester linkage involving a carboxyl group of aspartic or glutamic acid, or per- haps a C-terminal amino acid.

If a linkage through a serine or threonine hydroxyl group occurs, then the release of sugar would proceed through a base- catalyzed elimination reaction. Eliminations of this type are known and have been studied in model compounds consisting of glucose, and the methyl ester or the methyl amide of N-benzyl- oxycarbonylserine (19). In the cases which have been studied so far, the rate of hydrolysis of these compounds is appreciably slower than the rate of hydrolysis observed with the glucosyl- peptides. However, the unusual reactivity of the peptides could be due to participation of a neighboring group. So far no imidate esters of glucose have been described, to the best of our knowl-

edge. Model experiments have been reported which show that amide groups can participate in hydrolysis of glucosides and imidate esters are postulated to occur as intermediates in these reactions (20). For most known imidate esters, the rate constant of hydrolysis in acid is larger than the rate constants of hydrolysis in base. Examples are, however, known in which

hydrolysis at high pH is appreciably faster than at low pH (21). Insufficient evidence is currently available concerning the pH dependence of the hydrolysis of the glucosyl-peptide to either confirm or eliminate the possible involvement of an imidate ester. Finally, the formation of a glucosyl ester must be considered. The intermediate carbonium ion formed in the hydrolysis of the substrate could be stabilized by the participation of a neighboring carboxyl group. Participation of the carboxyl group has been invoked in the hydrolysis catalyzed by lysozyme and is supported by structural data obtained from x-ray analysis (22). This type of linkage is also implicated as an intermediate in the glucoside hydrolysis catalyzed by P-glucosidase (23). Glycosidic esters are known, and bond cleavage can occur by solvent attack on the ester carbonyl group as was observed with the glucosyl-peptides. Again, the rates of hydrolysis of model glycosidic esters is considerably slower than the rate of hydrolysis observed here (24, 25). However, as in the case of the serine linkages, the possible participation of a neighboring group may account for the high rate of hydrolysis. Therefore, at the present time, all types of linkages must be considered equally probable. Further experimentation is currently in progress to decide exactly what type of linkages are involved. Preliminary experiments, using the procedure of Koshland and Hoare (26)) strongly favor the involvement of a carboxyl group.

Aclcnowledgments-We wish to thank Merck Sharp and Dohme and Dr. Thomas H. Stout for providing, through their fermenta- tion facilities, the microorganisms used in these experiments.

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1. DOUDOROFF, M., BARKER, H. A., BND H.ISSID, W. Z., J. Biol. Chem., 168, 725 (1947).

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-LINKED COVALENT GLUCOSE-ENZYMEβPROPERTIES OF A The Mechanism of Action of Sucrose Phosphorylase: ISOLATION AND

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