CHARACTERISTICS OF CELLOBIOSE PHOSPHORYLASE

Transcription

1 CHARACTERISTICS OF CELLOBIOSE PHOSPHORYLASE JAMES K. ALEXANDER' Department of Botany and Bacteriology, Montana State College, Bozemian, Montana Received for publication November 22, 1960 The phosphorolysis of cellobiose was first demonstrated by Sih and McBee (1955a, b). These workers found that cellobiose phosphorylase was present in cell-free extracts of Clostridium thermocellum. Recently cellobiose phosphorylase has been reported from two other bacteria, Ruminococcus flavefaciens (Ayers, 1958, 1959), and Cellvibrio gilvus (Hulcher and King, 1958). This enzyme also catalyzes the synthesis of cellobiose froin a-d-glucose 1-phosphate and glucose (Sih, Nelson, and McBee, 1957; Ayers, 1959). In the present work, the stoichiometry of the reaction and the effect of physical and chemical agents on the activity of the enzyme were studied. Studies on the specificity of the enzyme revealed that D-glucose, D-xylose, L-xylose, 2-deoxyglucose, D-glucosamine, arsenate, and inorganic phosphate can act as substrates. Evidence has been obtained which indicates that the reaction proceeds by a single displacement mechanism. A preliminary report of this work has been published (Alexander, 1958). MATERIALS AND METHODS Glucose 6-phosphate, a-glucose 1-phosphate, 2-deoxyglucose, D-ribose, D-gluconate, A-glueonolactone, N-acetylglucosamine, D-glucosamine, L-arabinose, and lactose were obtained from Nutritional Biochemicals Corporation. Cellobiose was a product of Krishell Laboratories. D-Xylose, L-xylose, D-mannose, L-fructose, D- sorbitol, D-galactose, salicin, and trehalose were obtained from Difco Laboratories. Glucose was obtained from Allied Chemical and Dye Corporation; glueuronate from Sigma Chemical Company; gentiobiose from General Biochemicals Incorporated; and maltose from Pfanstiehl Chemical Company. 3-Glucose 1-phosphate was generously supplied by Dr. Charlotte Fitting. 1 Present address: Department of Biological Chemistry, Hahnemann Medical College, Philadelphia, Pennsylvania. Glucose oxidase was obtained from the Takamine Company. C. thermocellum strain 651 was used in this work. The organism was grown in a modified medium of McBee (1948) containing the following percentage composition: NaCl 0.3; (NH4)2- SO4, 0.1; K2HPO4, 0.05; KH2PO4, 0.05; MgSO4. 7H20, 0.01; CaCl2-2H20, 0.001; sodium thioglycolate, 0.02; yeast extract, 0.1; cellulose, 0.25; and NaHCO3, 0.5. To satisfy the anaerobic requirements of the organism, oxygen-free conditions were obtained by flushing the media with an inert gas (Hungate, 1950). Approximately 1 g (wet weight) of cells per liter was obtained in this medium. Cell-free extracts were prepared by grinding cells with glass beads in a Waring Blendor which was jacketed with an ice bath (Lamanna and Mallette, 1954). About 15 to 30 g of wet cells, suspended in 7 to 15 ml of water, and 20 to 40 g of glass beads were ground in a blender for 15 to 30 min. After grinding, 20 to 40 ml of water were added. Mixing was resumed until an even suspension was obtained and then the material was centrifuged for 1 hr at approximately 6,000 X g. The supernatant fluid served as the crude enzyme. The crude enzyme preparation was partially purified by (NH4)2S04 fractionation. To 8 ml of the crude preparation 3 ml of cold saturated (NH4)2S04 solution were added and this portion was allowed to stand 1 hr. After centrifugation at 6,000 X g for 1 hr the precipitate was discarded and 11 ml of saturated (NH4)2S04 solution were added to the supernatant. This portion was allowed to stand 1 hr and then it was centrifuged for 1 hr at 6,000 X g. The precipitate, containing cellobiose phosphorylase, was dissolved in water and diluted to 8 ml. This entire purification procedure was repeated two times. All steps in the purification were carried out at 5 C without adjusting the ph of the ammonium sulfate solution. Phosphoglucomutase, 903

2 904 ALEXANDER [VOL. 81 the only known interfering enzyme, was eliminated by this procedure (phosphoglucomutase activity was determined by measuring the disappearance in acid labile phosphate). Cellobiose or glucose 1-phosphatase activity could not be detected in either the crude or the purified preparations. Thus, the purified preparations could be used to study the cellobiose phosphorylase reaction in the absence of known interfering reactions. The purified enzyme preparations were used throughout this study. The ph of the reaction mixtures was 7.0; the incubation temperature 37 C. The reaction mixtures were buffered with barbital-acetate buffer (Michaelis, 1931). Analytical procedures. Cellobiose phosphorylase activity was determined by measuring the change in inorganic phosphate when the enzyme was incubated in the presence of either cellobiose and phosphate, or glucose and glucose 1-phosphate. Samples of the reaction mixtures were removed at various time intervals and the enzyme was inactivated by boiling for 3 min. After boiling, the protein was precipitated with either 5% trichloroacetic acid or N HCl. Phosphate was determined by the method of Fiske and SubbaRow (1925). Glucose 1- phosphate was estimated by measuring the increase in inorganic phosphate after hydrolysis in N HCI for 7 min at 100 C. Glucose was determined manometrically as described by Keilin and Hartree (1948). The crude glucose oxidase preparations used in the glucose determinations exhibited a small amount of f3-glucosidase activity. The error introduced in these determinations due to the hydrolysis of cellobiose was minimized by including two standards in each manometric analysis. One standard consisted of a known amount of glucose and the other contained glucose, cellobiose, glucose 1-phosphate, and phosphate in approximately the same amounts as the unknown sample. Cellobiose was hydrolyzed with,3-glucosidase and the increase in free glucose was determined manometrically. An active,b-glucosidase was precipitated from the crude glucose oxidase preparation with 50% acetone in the cold (a heat stable g-glucosidase inhibitor was recovered in the supernatant). The acetone-precipitated preparations retained glucose oxidase activity and hydrolyzed cellobiose about five times as fast as did the crude preparations. The hydrolysis of cellobiose proceeded at a slower rate than the oxidation of glucose. Extending the determination period to 3 to 4 hr, however, permitted complete hydrolysis of cellobiose. Standards similar to those described above were included in each analysis. Chromatography of sugars was carried out in the manner described by Sih et al. (1957). The amount of protein in the enzyme preparations was estimated by biuret method of Levin and Brauer (1951). RESULTS Stoichiometry and equilibrium of reaction. When cellobiose phosphorylase was incubated with cellobiose and phosphate, equimolar quantities of glucose and glucose 1-phosphate were formed (Table 1). The molar quantities of glucose and glucose 1-phosphate formed were approximately equal to the amounts of cellobiose and phosphate TABLE 1 Analysis of products and reactants phosphorylase reaction of cellobiose Expt No. 1 Expt No. 2 Expt No. 3 Initial Final Initial Final Initial Final,umoles/ml umoles/nl pimoles/ml Cellobiose Phosphate Glucose 1- phosphate Glucose K The reaction mixtures were incubated at 37 C. The ph of the reaction mixtures was 7.0. In experiment no. 1, the reaction mixture contained 4.0 ml enzyme (28 mg protein), 1.2 ml 0.1 M cellobiose, 0.8 ml 0.1 M phosphate, 2.0 ml 0.03 M barbital-acetate buffer, and was incubated for 80 min. In experiment no. 2, the reaction mixture contained 2.0 ml enzyme (20 mg protein), 0.8 ml M glucose 1-phosphate, 0.8 ml 0.1 M glucose, 4.0 ml 0.03 M barbital-acetate buffer, 0.4 ml water, and was incubated for 75 min. In experiment no. 3, the reaction mixture contained 1.0 ml enzyme (10 mg protein), 0.6 ml 0.1 M cellobiose, 0.6 ml 0.1 M phosphate, 0.2 ml M glucose 1-phosphate, 0.6 ml 0.1 M glucose, 1.0 ml 0.03 M barbitalacetate buffer, 4.0 ml water, and was incubated for 30 min.

3 19611 CELLOBIOSE PHOSPHORYLASE 905 utilized. When cellobiose phosphorylase was incubated with glucose and glucose 1-phosphate, the molar quantities of cellobiose and phosphate formed were equal to the molar quantities of reactants utilized. The equilibrium constant, K = (cellobiose)- (phosphate)/(glucose)(glucose 1-phosphate), is about 4.3 (Table 1). In these experiments, the activity was followed by measuring the change in phosphate at 10- to 20-min intervals, until no further change was detected. Since nearly the same values were obtained from either side, it appears that these values approximate the true equilibrium constant of the reaction. Effect of hydrogen ion concentration. The optimal ph for the activity of cellobiose phosphorylase is about 7.0, in M barbital-acetate buffer. The enzyme is active over quite a wide range; activity has been detected at ph 4.6 and at ph 8.1. No activity was observed, however, at ph 4.1 or at ph 8.7. TABLE 2 Effect of inhibitors on the activity phosphorylase of cellobiose Addition Per Cent Inhibition p-chloromercuribenzoate, 1 X 10-M.42 p-chloromercuribenzoate, 2 X 10l- 100 p-chloromercuribenzoate, 1 X 10-6 M; cysteine, 1 X 10-4 M... 7 p-chloromercuribenzoate, 2 X 10-5 M; cysteine, 1 X 10-4 M AgNO3, 1 X AgNO3, 1 X 10-4 M; cysteine, 1 X 104 M. 91 AgNO3, 1 X 104 M; cysteine, 1 X 10-2 M.. 0 Phlorizin, 1.25 X 10-3 M... 8 Phlorizin, 2.5 X 10-3 M Phlorizin, 5.0 X 103 M NaF, 0.1 M 21 NaF, The reaction mixtures were incubated at 37 C for 1 hr. Each of the reaction mixtures contained 0.5 ml enzyme (4 mg protein), 0.3 ml 0.1 M cellobiose, 0.2 ml 0.1 M phosphate, 0.2 ml 0.03 M barbital-acetate buffer. The total volume was made up to 2.0 ml with water, cysteine (0.02 ml (0.1 M) or 0.2 ml (0.001 M)), or inhibitor. The amounts of the various inhibitors added were: 0.2 or 0.4 ml 1 X 10-4 M p-chloromercuribenzoate; 0.2 ml M AgNO3; 0.2, 0.4, or 0.8 ml M phlorizin; and 0.4 or 0.8 ml 0.5 M NaF. TABLE 3 Effect of glucose on cellobiose phosphorolysis Cellobiose Additions Glucose Phosphate Esterified Per Cent Inhibition jsmoles/ml pmoles/ml The reaction mixtures were incubated for 20 min at 37 C. The reaction mixtures contained 0.2 ml enzyme (3 mg protein); 0.075, 0.15, or 0.3 ml 0.1 M cellobiose; ml 0.1 M phosphate; or 0.15 ml 0.1 M glucose; ml 0.01 M ethylenediamine-tetraacetic acid (EDTA), and were diluted to 1.0 ml with water. EDTA was used in certain experiments after it was learned that the enzyme was sensitive to sulfhydryl reagents. Since no increased activity was observed with EDTA, it was omitted from some of the experiments. Inhibitors. Cellobiose phosphorylase activity was completely inhibited by 2 X 10-5 M p- chloromercuribenzoate, whereas 1 X 10-5 M p-chloromercuribenzoate caused about 40% inhibition (Table 2). The inhibition could be partially reversed by 1 X 10-4 M cysteine. Silver nitrate (1 X 10-4 M) also inhibited cellobiose phosphorylase activity. This inhibition was completely reversed by 1 X 102 M cysteine. These data suggest that the enzyme has an essential sulfhydryl group. Inhibition of cellobiose phosphorylase by phlorizin was directly proportional to the concentration of phlorizin between and M. About 20% of the activity is inhibited by 0.1 M NaF, whereas 0.2 M NaF almost completely inhibited activity. The same concentrations (0.1 and 0.2 M) of NaCl or (NH4)2SO4 caused little or no inhibition. Magnesium sulfate in concentrations of 0.01 or 0.02 M had no significant effect on the activity. The inhibition of phosphorolysis by glucose is shown in Table 3. Further study will be needed to determine the nature of glucose inhibition.

4 906 ALEXANDER [VOL. 81 Specificity of cellobiose phosphorylase. When cellobiose phosphorylase was incubated with glucose 1-phosphate and D-xylose, L-xylose, D-glucosamine, 2-deoxyglucose, or glucose, phosphate was liberated as shown in Table 4. The liberation of phospnate suggests that di- TABLE 4 Liberation of inorganic phosphate when cellobiose phosphorylase was incubated with glucose 1-phosphate and various other compounds Expt No. Compound Added Phosphate Liberated I hr 2 hr Amoles/ml 1 None 0 D-Glucose D-Xylose L-Xylose Deoxyglucose D-Gluconate Glucuronate Glucose 6-phosphate D-Glucose D-Ribose D-Sorbitol D-Glucose A-gluconolactone D-Glucosamine N-Acetylglucosamine D-Glucose L-Arabinose 0 0 L-Fructose 0 0 D-Galactose 0 0 D-Mannose The reaction mixtures contained 0.5 ml enzyme (6 to 8 mg protein) 0.3 ml 0.1 M monosaccharide, 0.2 ml M glucose 1-phosphate, 0.2 ml 0.01 M EDTA, and 0.8 ml water. saccharides are synthesized according to reactions 1 to 5 (see below). The identity of these compounds has not been proved. The argument for suggesting a 3-1,2- glucosidic linkage in the L-xylose-containing disaccharide is presented below. Paper chromatograms of the reaction mixtures containing D-xylose or L-xylose likewise suggest that pentose-containing disaccharides had been synthesized (Fig. 1). After the chromatograms were dipped into an aniline-phthalic acid color developer and allowed to stand at room temperature, red spots developed at the positions of the pentoses and the presumed pentose-containing disaccharides, but no color was present in the cellobiose position. Cellobiose did not develop a color until after the chromatograms were heated. The color of the pentoses and pentosecontaining disaccharides turned reddish-brown upon heating, in contrast to the plain brown color of cellobiose. No attempts were made to obtain chromatographic separation of the disaccharides synthesized with D-glucosamine or 2- deoxyglucose. No significant amount of phosphate was liberated when cellobiose phosphorylase was incubated with glucose 1-phosphate and D- gluconate, D-glucuronate, D-glucose 6-phosphate, D-ribose, D-sorbitol, 6-gluconolactone, N-acetylglucosamine, L-arabinose, L-fructose, D-galactose, or D-mannose (Table 4). These compounds apparently are unable to act as glucosyl acceptors. Phosphorolysis of disaccharides. Cellobiosephosphorylase was unable to catalyze the phosphorolysis of gentiobiose, salicin, lactose, or maltose, for no significant amount of phosphate disappeared when the enzyme was incubated with phosphate and these sugars. In contrast to the results with these sugars, relatively large amounts of phosphate disappeared when the enzyme was incubated with cellobiose and phosphate. The enzyme presumably would D-Glucose + glucose 1 phosphate = 4-0-,3-D-glucopyranosyl-D-glucopyranose (cellobiose) + phosphate (1) D-Xylose + glucose 1 phosphate = D-glucopyranosyl-D-xylopyranose + phosphate (2) L-Xylose + glucose 1 phosphate = 2-0-f3-D-glucopyranosyl-L-xylopyranose + phosphate (3) 2-Deoxyglucose + glucose 1 phosphate = 4-O-$-D-glucopyranosyl-D-deoxyglucose + phosphate (4) D-Glucosamine + glucose 1 phosphate -, 4-0-,B-D-glucopyranosyl-D-glucosamine + phosphate (5)

5 w... ^....P': :;.0. :.. '' : 1961] CELLOBIOSE PHOSPHORYLASE 907 that an inversion occurred in the reaction (Sih et al., 1957; Ayers, 1959). The occurrence of an inversion is further supported by the finding that the enzyme is specific for a-glucose 1- phosphate and it is unable to act on,8-glucose 1-phosphate as shown in Table 5. - Arsenolysis of cellobiose. Arsenate is able to substitute for phosphate in the reaction resulting A i. in the arsenolysis of cellobiose. When the enzyme *f::* *.: was incubated with 100,umoles of cellobiose and 10,umoles of arsenate, nearly 46,umoles of :.:<.;. glucose were formed (Table 6). Since the amount of glucose formed was more than 4 times as great as the amount of arsenate present, it is apparent that the enzyme catalyzed the arsenolysis of cellobiose....i. Mechanism of the reaction. The occurrence of an inversion in both the cellobiose and maltose phosphorylase (Fitting and Doudoroff, 1952) reactions suggests that the mechanism of these reactions is similar. Additional information on A_:F- s the mechanism of the cellobiose phosphorylase reaction is shown in the following experiments. An experiment was designed to determine the ability of cellobiose phosphorylase to catalyze the exchange between glucose 1-phosphate and arsenate. If this exchange occurred, glucose 1- arsenate would be formed and this product would be expected to undergo spontaneous decomposition, resulting in the formation of free glucose and arsenate. No significant amount of free glucose was formed when the enzyme was incubated with glucose 1-phosphate and arsenate :.: D... ^ a c D Fig. 1. Chromatogram on the left shows a compound tentatively identified as 4-0-#-Dglucopyranosyl-D-xylopyranose in the lower spots of rows A and B. Row A is from 0.2 ml of the reaction mixture described in Table 4; row B is from 0.1 ml of the same reaction mixture. Row C shows cellobiose and row D shows D-xylose. The chromatogram on the right is the same as the other chromatogram except that L-xylose was used instead of D-xylose. The solvent was applied to the paper for about 20 hr at room temperature. catalyze the phosphorolysis of the four new compounds which appear to have been synthesized. f-d-glucose 1-phosphate. The synthesis of cellobiose from a-glucose 1-phosphate and glucose with cellobiose phosphorylase suggested TABLE 5 Liberation of phosphate when cellobiose phosphorylase was incubated with glucose and,- or a- glucose 1 -phosphate Reaction Mixtures Compound Added Phosphate ~~~~~~~Liberated 30 min 60 miii,umoles/ml 1 a-glucose 1-phosphate ,B-Glucose 1-phosphate (boiled) f,-glucose 1-phosphate The reaction mixtures contained 0.15 ml enzyme (2 mg protein), 0.2 ml 0.1 M glucose, 0.08 ml M a-glucose 1-phosphate or 0.6 ml 0.01 M,B-glucose 1-phosphate, 0.05 ml 0.14 M barbitalacetate buffer and were diluted to 1.0 ml with water.

6 9708 ALEXANDER [VOL. 81 TABLE 6 Arsenolysis of cellobiose and the lack of exchange between glucose 1-phosphate and arsenate Reaction Mixture* No. with Substrate Addition Hus GlucoseGlcs Cello- Cellobiose Cellobiose Gluhose 1-phosbiose arsenate phosphate phates- phate arsenate,moles glucose formed/ml Reaction mixtures 1, 2, and 3 contained 1.0 ml 0.1 M cellobiose; reaction mixtures 2 and 5 contained 0.1 ml 0.1 M arsenate; reaction mixture 3 contained 0.1 ml 0.1 M phosphate; reaction mixtures 4 and 5 contained 1.0 ml M glucose 1- phosphate; each reaction mixture contained 0.5 ml enzyme (5 mg protein) and 0.4 ml of 0.03 M barbital-acetate buffer (total volume 2.0 ml). The reaction mixtures were buffered with barbital-acetate buffer. * In reaction mixtures 1, 2, and 3, glucose was determined manometrically. In reaction mixtures 4 and 5, glucose was determined by the method of Saifer, Valenstein, and Hughes (1941). (Table 6). It has been shown that arsenate is able to substitute for phosphate in the decomposition of cellobiose. Therefore, it might be inferred that the exchange between glucose 1-phosphate and phosphate does not occur because the exchange between glucose 1-phosphate and arsenate does not occur. The following experiment was designed to determine the ability of cellobiose phosphorylase to catalyze the exchange between cellobiose and and D-xylose. If this exchange occurred, 4-0-,B-D-glucopyranosyl-D-xylopyranose and free glucose would be expected to be formed in the absence of phosphate. No spot corresponding to 4-0-/3-D-glucopyranosyl-D-xylopyranose2 was present when this reaction mixture was chromatographed i-D-Glucopyranosyl -D-xylopyranose could be distinguished from cellobiose in that it formed a red color with the aniline-phthalic acid color developer. As a control, another reaction mixture was included in this experiment which differed from the above in that it contained a small amount of phosphate. The following reactions were expected to occur in this reaction mixture: Cellobiose + phosphate glucose 1-phosphate + glucose D-xylose + glucose 1-phosphate = Dglucopyranosyl-D-xylopyranose + phosphate The phosphate liberated in the synthesis of 4-0- g-d-glucopyranosyl-d-xylopyranose would be able to react again in the phosphorolysis of cellobiose. The detection of a compound giving the reaction expected of the pentose-containing disaccharide in the chromatogram is evidence that these reactions did occur. The results indicate that 4-0-,3-D-glucopyranosyl-D-xylopyranose is formed only in the presence of phosphate. It appears, therefore, that the direct exchange of cellobiose and D-xylose is not catalyzed by cellobiose phosphorylase. Likewise, the enzyme would not be expected to catalyze the exchange between cellobiose and glucose. The inability of cellobiose phosphorylase to catalyze the exchange reactions between glucose 1-phosphate and arsenate, and cellobiose and D- xylose is analogous to the results obtained with maltose phosphorylase (Fitting and Doudoroff, 1952). It seems likely, therefore, that the cellobiose and maltose phosphorylase reactions have similar mechanisms. DISCUSSION Thermodynamic considerations. On the basis of the equilibrium constant of the cellobiose phosphorylase reaction the free energy change for the synthesis of cellobiose from glucose and glucose 1-phosphate is about -900 cal. The free energy of hydrolysis of cellobiose is estimated to be -3,900 cal. The equilibrium of the maltose phosphorylase reaction (Fitting and Doudoroff, 1952) appears to be identical to the equilibrium of the cellobiose phosphorylase reaction. From the equilibrium of the maltose phosphorylase reaction the free energy of hydrolysis of maltose has been estimated to be about -4,000 cal (Kalckar, 1954). For this value the energy of,b-glucose 1-phosphate, the product of maltose phosphorolysis, was assumed to be nearly the same as the energy of a-glucose 1-phosphate. This assumption

7 19611 CELLOBIOSE PHOSPHORYLASE 909 is supported by the information obtained in this study. Any significant influence by the configuration of the phosphate or glucosidic bonds on the energy level should be reflected in the equilibrium constants of these phosphorylase reactions. It seems likely, therefore, that the configuration of these bonds has little or no influence on the energy level. Specificity of cellobiose phosphorylase. The ability of cellobiose phosphorylase to catalyze the condensation of glucose 1-phosphate with D-glucose, D-xylose, 2-deoxyglucose, or glucosamine can be explained by assuming that the enzyme is relatively nonspecific for the C-2 and C-5 groups of these substrates. It seems reasonable to assume further that these compounds are attached to a glucosyl radical through a f3-1, 4-glucosidic linkage as in cellobiose. On the basis of these assumptions, certain interpretations can be made with regard to the specificity of cellobiose phosphorylase. The H and OH groups of C-2 cannot be reversed because mannose did not react. However, 2-deoxyglucose did react, although the reaction was not as rapid as with glucose. Thus, the OH group cannot replace the H group of C-2, but the OH group of C-2 can be replaced by H. Likewise, this OH group can be replaced by NH2 for glucosamine was able to react. The lack of specificity at this position is limited, however, for the OH group could not be replaced by the acetylamine group of N-acetylglucosamine. The inability of D-ribose to act as a glucosyl acceptor suggests that the H and OH groups attached to C-3 cannot be reversed. Likewise, the position of the H and OH groups attached to C-4 cannot be reversed for D-galactose did not react. The ability of both D-glucose and D-xylose to react indicates that CH20H group can be replaced by an H group. Therefore, the enzyme appears to be somewhat nonspecific for the groups attached to C-5. In view of these specific requirements of the enzyme, it was surprising to find that L-xylose was able to act as a glucosyl acceptor. L-Xylose would not be expected to combine with a glucosyl group through C-4 because the H and OH groups on C-2, C-3, and C-4 would be reversed from the position in which they occur in other compounds which react. If, however, L-xylose were to attach to the glucosyl group through C-2 in such a way that C-4 of L-xylose would occupy the same position as C-2 of glucose, then the sequence of H and OH groups in L-xylose would be the same as in D-glucose. Thus, on the basis of these data a 3-1,2-glucosidic linkage in the L- xylose-containing disaccharide would seem possible. Mechanism of reaction. The occurrence of an inversion in the cellobiose phosphorylase reaction and the inability of the enzyme to catalyze the exchange between either cellobiose and D-xylose or glucose 1-phosphate and arsenate indicates a single displacement mechanism for the reaction as described by Koshland (1954a, b). Koshland's discussion of the mechanism of the maltose phosphorylase reaction apparently can be extended to the cellobiose phosphorylase reaction. The only significant difference in maltose and cellobiose phosphorylase appears to be the specificity for the configuration of the glucosidic and phosphoglucose bonds. That the active surface of the two enzymes is similar is suggested by the similarity in specificity for various substrates. D-glucose, D-xylose, phosphate, and arsenate were able to act as substrates for both enzymes, whereas D-mannose-, D-ribose, D- galactose, L-arabinose, and D-gluconate were inactive (Fitting and Doudoroff, 1952). It seems possible that the active surface of cellobiose phosphorylase is essentially a mirror image of the surface of maltose phosphorylase. ACKNOWLEDGMENTS The author wishes to thank R. H. McBee and N. M. Nelson for their advice in this work. This investigation was supported in part by a research grant (A-1088) from the National Institute of Arthritis and Metabolic Diseases. SUMMARY Cellobiose phosphorylase, obtained from cellfree extracts of Clostridium thermocellum, was purified sufficiently to remove known interfering enzymes. The enzyme catalyzes the phosphorolysis of cellobiose with the formation of equimolar quantitites of glucose and glucose 1-phosphate. The reaction is reversible, for equimolar quantities of cellobiose and inorganic phosphate are formed from glucose and a-d-glucose 1-phosphate. The equilibrium constant, K = (cellobiose) (phosphate)/(glucose)(glucose 1-phosphate), at ph 7 and 37 C, is about 4.3.

8 '910 ALEXANDER [VOL. 81 D-Glucose, D-xylose, L-xylose, 2-deoxyglucose, D-glucosamine, phosphate, and arsenate can serve as substrates for the enzyme. (-Glucose 1- phosphate was unable to replace a-glucose 1- phosphate indicating that an inversion occurs in the reaction. On the basis of the inversion and the inability of the enzyme to catalyze the exchange between either cellobiose and D-xylose or glucose 1-phosphate and arsenate, a single displacement mechanism is proposed for the reaction. REFERENCES ALEXANDER, J. K Studies on a purified cellobiose phosphorylase. Bacteriol. Proc., 1958, 125. AYERS, W. A Phosphorylation of cellobiose and glucose by Ruminococcus flave faciens. J. Bacteriol., 76, AYERS, W. A Phosphorolysis and synthesis of cellobiose by cell extracts from Ruminococcus flavefaciens. J. Biol. Chem., 233, FISKE, C. H., AND Y. SUBBARow 1925 The colorimetric determination of phosphorus. J. Biol. Chem., 66, FIrrING, C., AND M. DOUDOROFF 1952 Phosphorolysis of maltose by enzyme preparations from Neisseria meningitidis. J. Biol. Chem., 199, HU1,CHER, F. H., AND K. W. KING 1958 Metabolic basis for disaccharide preference in a Cellvibrio. J. Bacteriol. 76, HUNGATE, R. E The anaerobic mesophilic cellulolytic bacteria. Bacteriol. Rev., 14, KALCKAR, H. M The mechanism of transglycosidation. In The mechanism of enzyme action, pp Edited by W. D. McElroy and B. Glass. The Johns Hopkins Press, Baltimore. KEILIN, D., AND E. F. HARTREE 1948 The use of glucose oxidase (notatin) for the determination of glucose in biological material and for the study of glucose-producing systems by manometric methods. Biochem. J., 42, KOSHLAND, D. E. 1954a Group transfer as a substitution mechanism. In The mechanism of enzyme action, pp Edited by W. D. McElroy and B. Glass. The Johns Hopkins Press, Baltimore. KOSHLAND, D. E. 1954b A physical organic approach to enzymatic mechanisms. Trans. N. Y. Acad. Sci., 16, LAMANNA, C., AND M. F. MALLETTE 1954 Use of glass beads for the mechanical rupture of microorganisms in concentrated suspensions. J. Bacteriol., 67, LEVIN, R., AND R. W. BRAUER 1951 The Biuret reaction for determiination of proteins-an improved reagent and its application. J. Lab. Clin. Med., 38, McBEE, R. H The culture and physiology of a thermophilic cellulose-fermenting bacterium. J. Bacteriol., 56, MICHAELIS, L Der acetat-veronal puffer. Biochem. Z., 234, SAIFER, A., F. VALENSTEIN, AND J. P. HUGHES 1941 The photometric determination of sugar in biological fluids by ferricyanide reduction. J. Lab. Clin. Med., 26, SIH, C. J., AND R. H. McBEE 1955a A phosphorylase active on cellobiose. Bacteriol. Proc., 1955, 126. SIH, C. J., AND R. H. McBEE 1955b A cellobiose phosphorylase in Clostridium thermocellum. Proc. Montana Acad. Sci., 15, SIH, C. J., N. M. NELSON, AND R. H. McBEE 1957 Biological synthesis of cellobiose. Science, 126,