J. Biochem. 112, .40-44 (1992)
Synthetic Reaction of Cellvibrio gilvus Cellobiose Phosphorylase Motomitsu Kitaoka,* Takashi Sasaki," and Hajime Taniguchi** 'Nippon Petrochemicals Co., Ltd., Tsukuba, Ibaraki 300-26; and "National Food Research Institute, Tsukuba, Ibaraki 305 Received for publication, January 13, 1992
The synthetic reactions of the cellobiose phosphorylase from Cellvibrio gilvus were investigated in detail. It was found that, besides D-glucose, some sugars having substitution or deletion of the hydroxyl group at C2 or C6 of the D-glucose molecule could serve as a glucosyl acceptor, though less effectively than D-glucose. The enzyme showed higher activity with £-D-glucose than with the a-anomer as an acceptor. This result indicates that it recognizes the anomeric hydroxyl group not involved directly in the reaction, fiD-Cellobiose was also phosphorolyzed faster than the a-anomer. Substrate inhibition was observed with D-glucose, 6-deoxy-D-glucose, or D-glucosamine as an acceptor, with D-glucose being most inhibiting. This inhibition was studied in detail and it was found that D-glucose competes with a-D-glucose-1-phosphate for its binding site. A model of competitive substrate inhibition was proposed, and the experimental datafitwell to the theoretical values that were calculated in accordance with this model.
Cellobiose phosphorylase [EC 2.4.1.20] is one of the enzymes phosphorolyzing glucosides. It catalyzes reversible phosphorolysis of D-cellobiose into D-glucose and aD-glucose-1-phosphate (G-l-P) with inversion of the anomeric configuration. It is present in Clostridium thermocellum (1), Riuninococcus flavefaciens (2), Cellvibrio gilvus (3), Fomes annosus (4), and Cellulomonas (5, 6). Alexander partially purified the enzyme from C. thermocellum (7) and synthesized several disaccharides from G-l-P and acceptor sugars using the enzyme preparation (8). We reported a convenient synthetic method for D-glucosylD-xylose from G-l-P and D-xylose using C. gilvus cells as an immobilized cellobiose phosphorylase (9). We purified the enzyme from C. gilvus to an electrophoretically homogeneous state and reported its properties (10). We also found that its reaction proceeded through an ordered bi bi mechanism {11). In the present paper, an extensive kinetic study on the synthetic reaction of this enzyme is reported. MATERIALS AND METHODS Materials—a-D-Glucose-1 -phosphate (G-l-P) dipotassium salt, /9-D-glucose, and /?-D-cellobiose were purchased from Sigma (St. Louis, USA). ar-D-Cellobiose (containing about 5% of the /9-anomer) was obtained by ethanol precipitation from a D-cellobiose solution. All other chemicals used in the experiments were of reagent grade. Cellobiose phosphorylase was purified from C. gilvus cells by the method described by Kitaoka et al. (11). Assay Methods—Reactions of the cellobiose phosphorylase were carried out at 37'C in 50 mM Tris-HCl buffer (pH 7.0) containing 5 mM MgCl2 and 0.02% bovine serum albumin as a stabilizer of the enzyme. One unit of the activity was defined as the amount of the enzyme which produces 1 /x mol of D-glucose or G-1 -P per min with 10 mM D-cellobiose and 10 mM inorganic phosphate (P() under the above conditions. The initial rate of the phosphorolysis was assayed by measuring the amount of G-l-P or D-glucose 40 Downloaded from https://academic.oup.com/jb/article-abstract/112/1/40/837926 by Insead user on 05 May 2018
formed during the enzymatic reaction. That of the synthetic reaction was determined by measuring the amount of Pi liberated from G-l-P with an acceptor. The amount of G-l-P was measured by using the phosphoglucomutase-glucose-6-phosphate dehydrogenase system (12). D-Glucose was measured by means of the glucose oxidase-peroxidase method with mutarotase (13) using the Glucose C Test Wako (Wako Pure Chemicals, Osaka). P, in the presence of G-l-P was measured selectively by the method of Lowry and Lopez (14). Kinetic Parameters—Kinetic parameters were calculated from the experimental results following the GaussNewton method described by Cleland (15) using computer programs written in BASIC. RESULTS AND DISCUSSION Substrate Specificity of the Synthetic Reaction—Table I indicates relative initial velocities obtained with various sugars as a glucosyl acceptor. Derivatives at Cl of the D-glucose molecule such as methyl-D-glucosides and 1,5-anhydro-D-glucitol did not serve as an acceptor. Isomers at C3 (D-allose), C4 (D-galactose), and C5 (L-idose) did not serve as an acceptor. In contrast, some of the C2 derivatives (D-mannose, 2-deoxy-D-glucose, and D-glucosamine) and C6 ones (6-deoxy-D-glucose and D-xylose) could act as an acceptor, although less effectively than D-glucose. These results indicate that the configurations of the D-glucose molecule at Cl, C3, C4, and C5 are strictly required. Apart from D-glucose derivatives, ketose, pentose (except D-xylose), D-glucono-tf-lactone, and sugar alcohols had no acceptor activity. Essentially the same specificity for the acceptor molecule was reported with the partially purified enzyme from C. thermocellum (7). The acceptor specificity for the C2 derivatives is significantly different from that reported for a maltose phosphorylase (16). The cellobiose phosphorylase can accept an axial hydroxyl group at C2 (D-mannose) whereas it cannot J. Biochem.
41
Synthetic Reaction of Cellobiose Phosphorylase accept bulky substitution at the C2 hydroxyl group (iV-acetyl-D-glucosamine). On the other hand, the maltose phosphorylase cannot accept the former whereas it can accept the latter. Furthermore, the deletion of the C2 hydroxyl group (2-deoxy-D-glucose) or substitution with an amino group (D-glucosamine) resulted in a significant decrease in the initial rate in the case of cellobiose phosphorylase. In
the case of the maltose phosphorylase, deletion (2-deoxyD-glucose), substitution with an amino group (D-glucosamine) or substitution with a bulky group (iV-acetylD-glucosamine) did not decrease the initial rates. Cellobiose phosphorylase, therefore, must have a recognition site for
TABLE I. Substrate specificity in the synthetic reaction. Values are indicated as //mol/min-U . For experimental details, see the text. —, under 0.03. Substrate Substrate D-Fructose D-Glucose 1.37 — a -Methyl-D-glucoside L-Sorbose — — D-Lyxose — P -Methyl- D-glucoside D-Ribose — 1,5-Anhydro-D-glucitol L-Arabinose — D-Mannoee 0.054 D-Arabinose — 2-Deoxy-D-glucose 0.034 L-Xylose — D-Glucosamine 0.12 — iV-Acetyl-D-glucosamine myo-Inositol — _ D-Glucono-cJ-lactone — D-Allose — D-Galactose D-Glucitol — L-Idose D-Mannitol — 6-Deoxy-D-glucose 0.70 — D-Glucuronic acrid D-Xylose 0.055
TABLE II. Apparent kinetic parameters of various substrates. D-Glucose* D-Mannose" 2-Deoxy-D-gluco8eb D-Glucosaminec 6-Deoxy-D-glucose" D-Xylose"
(mM) 2.1 115 168 13
(^mol/min-U)
1.7 0.68 0.64 0.26 24 2.4 84 0.54 Values were calculated at the following concentrations: "2-10 mM, "5-100 mM, c5-20 mM, and "2.5-50 mM. concentration (mM) Fig. 2. The v-[a] plots of the various substrates. • , D-glucose; O, 6-deoxy-D-glucose; A , D-mannose; A , 2-deoxy-D-glucose; • , D-glucosamine; D,D-xylose. Solid lines are calculated curves using the Michaelis-Menten equation.
.S 0.25 o o o
time (min) Fig. 1. Time course of the reactions with the substrates of both anomeric types. O,
Synthetic Reaction of Cellvibrio gilvus Cellobiose Phosphorylase Motomitsu Kitaoka,* Takashi Sasaki," and Hajime Taniguchi** 'Nippon Petrochemicals Co., Ltd., Tsukuba, Ibaraki 300-26; and "National Food Research Institute, Tsukuba, Ibaraki 305 Received for publication, January 13, 1992
The synthetic reactions of the cellobiose phosphorylase from Cellvibrio gilvus were investigated in detail. It was found that, besides D-glucose, some sugars having substitution or deletion of the hydroxyl group at C2 or C6 of the D-glucose molecule could serve as a glucosyl acceptor, though less effectively than D-glucose. The enzyme showed higher activity with £-D-glucose than with the a-anomer as an acceptor. This result indicates that it recognizes the anomeric hydroxyl group not involved directly in the reaction, fiD-Cellobiose was also phosphorolyzed faster than the a-anomer. Substrate inhibition was observed with D-glucose, 6-deoxy-D-glucose, or D-glucosamine as an acceptor, with D-glucose being most inhibiting. This inhibition was studied in detail and it was found that D-glucose competes with a-D-glucose-1-phosphate for its binding site. A model of competitive substrate inhibition was proposed, and the experimental datafitwell to the theoretical values that were calculated in accordance with this model.
Cellobiose phosphorylase [EC 2.4.1.20] is one of the enzymes phosphorolyzing glucosides. It catalyzes reversible phosphorolysis of D-cellobiose into D-glucose and aD-glucose-1-phosphate (G-l-P) with inversion of the anomeric configuration. It is present in Clostridium thermocellum (1), Riuninococcus flavefaciens (2), Cellvibrio gilvus (3), Fomes annosus (4), and Cellulomonas (5, 6). Alexander partially purified the enzyme from C. thermocellum (7) and synthesized several disaccharides from G-l-P and acceptor sugars using the enzyme preparation (8). We reported a convenient synthetic method for D-glucosylD-xylose from G-l-P and D-xylose using C. gilvus cells as an immobilized cellobiose phosphorylase (9). We purified the enzyme from C. gilvus to an electrophoretically homogeneous state and reported its properties (10). We also found that its reaction proceeded through an ordered bi bi mechanism {11). In the present paper, an extensive kinetic study on the synthetic reaction of this enzyme is reported. MATERIALS AND METHODS Materials—a-D-Glucose-1 -phosphate (G-l-P) dipotassium salt, /9-D-glucose, and /?-D-cellobiose were purchased from Sigma (St. Louis, USA). ar-D-Cellobiose (containing about 5% of the /9-anomer) was obtained by ethanol precipitation from a D-cellobiose solution. All other chemicals used in the experiments were of reagent grade. Cellobiose phosphorylase was purified from C. gilvus cells by the method described by Kitaoka et al. (11). Assay Methods—Reactions of the cellobiose phosphorylase were carried out at 37'C in 50 mM Tris-HCl buffer (pH 7.0) containing 5 mM MgCl2 and 0.02% bovine serum albumin as a stabilizer of the enzyme. One unit of the activity was defined as the amount of the enzyme which produces 1 /x mol of D-glucose or G-1 -P per min with 10 mM D-cellobiose and 10 mM inorganic phosphate (P() under the above conditions. The initial rate of the phosphorolysis was assayed by measuring the amount of G-l-P or D-glucose 40 Downloaded from https://academic.oup.com/jb/article-abstract/112/1/40/837926 by Insead user on 05 May 2018
formed during the enzymatic reaction. That of the synthetic reaction was determined by measuring the amount of Pi liberated from G-l-P with an acceptor. The amount of G-l-P was measured by using the phosphoglucomutase-glucose-6-phosphate dehydrogenase system (12). D-Glucose was measured by means of the glucose oxidase-peroxidase method with mutarotase (13) using the Glucose C Test Wako (Wako Pure Chemicals, Osaka). P, in the presence of G-l-P was measured selectively by the method of Lowry and Lopez (14). Kinetic Parameters—Kinetic parameters were calculated from the experimental results following the GaussNewton method described by Cleland (15) using computer programs written in BASIC. RESULTS AND DISCUSSION Substrate Specificity of the Synthetic Reaction—Table I indicates relative initial velocities obtained with various sugars as a glucosyl acceptor. Derivatives at Cl of the D-glucose molecule such as methyl-D-glucosides and 1,5-anhydro-D-glucitol did not serve as an acceptor. Isomers at C3 (D-allose), C4 (D-galactose), and C5 (L-idose) did not serve as an acceptor. In contrast, some of the C2 derivatives (D-mannose, 2-deoxy-D-glucose, and D-glucosamine) and C6 ones (6-deoxy-D-glucose and D-xylose) could act as an acceptor, although less effectively than D-glucose. These results indicate that the configurations of the D-glucose molecule at Cl, C3, C4, and C5 are strictly required. Apart from D-glucose derivatives, ketose, pentose (except D-xylose), D-glucono-tf-lactone, and sugar alcohols had no acceptor activity. Essentially the same specificity for the acceptor molecule was reported with the partially purified enzyme from C. thermocellum (7). The acceptor specificity for the C2 derivatives is significantly different from that reported for a maltose phosphorylase (16). The cellobiose phosphorylase can accept an axial hydroxyl group at C2 (D-mannose) whereas it cannot J. Biochem.
41
Synthetic Reaction of Cellobiose Phosphorylase accept bulky substitution at the C2 hydroxyl group (iV-acetyl-D-glucosamine). On the other hand, the maltose phosphorylase cannot accept the former whereas it can accept the latter. Furthermore, the deletion of the C2 hydroxyl group (2-deoxy-D-glucose) or substitution with an amino group (D-glucosamine) resulted in a significant decrease in the initial rate in the case of cellobiose phosphorylase. In
the case of the maltose phosphorylase, deletion (2-deoxyD-glucose), substitution with an amino group (D-glucosamine) or substitution with a bulky group (iV-acetylD-glucosamine) did not decrease the initial rates. Cellobiose phosphorylase, therefore, must have a recognition site for
TABLE I. Substrate specificity in the synthetic reaction. Values are indicated as //mol/min-U . For experimental details, see the text. —, under 0.03. Substrate Substrate D-Fructose D-Glucose 1.37 — a -Methyl-D-glucoside L-Sorbose — — D-Lyxose — P -Methyl- D-glucoside D-Ribose — 1,5-Anhydro-D-glucitol L-Arabinose — D-Mannoee 0.054 D-Arabinose — 2-Deoxy-D-glucose 0.034 L-Xylose — D-Glucosamine 0.12 — iV-Acetyl-D-glucosamine myo-Inositol — _ D-Glucono-cJ-lactone — D-Allose — D-Galactose D-Glucitol — L-Idose D-Mannitol — 6-Deoxy-D-glucose 0.70 — D-Glucuronic acrid D-Xylose 0.055
TABLE II. Apparent kinetic parameters of various substrates. D-Glucose* D-Mannose" 2-Deoxy-D-gluco8eb D-Glucosaminec 6-Deoxy-D-glucose" D-Xylose"
(mM) 2.1 115 168 13
(^mol/min-U)
1.7 0.68 0.64 0.26 24 2.4 84 0.54 Values were calculated at the following concentrations: "2-10 mM, "5-100 mM, c5-20 mM, and "2.5-50 mM. concentration (mM) Fig. 2. The v-[a] plots of the various substrates. • , D-glucose; O, 6-deoxy-D-glucose; A , D-mannose; A , 2-deoxy-D-glucose; • , D-glucosamine; D,D-xylose. Solid lines are calculated curves using the Michaelis-Menten equation.
.S 0.25 o o o
time (min) Fig. 1. Time course of the reactions with the substrates of both anomeric types. O,
