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Synthetic Reaction of Cellvibrio Gilvus Cellobiose Phosphorylase

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Synthetic Reaction of Cellvibrio Gilvus Cellobiose Phosphorylase J. Biochem. 112,,40-44 (1992) Synthetic Reaction of Cellvibrio gilvus Cellobiose Phosphorylase Motomitsu Kitaoka,* Takashi Sasaki,** and Haj ime 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 6-D-glucose than with the ƒ¿-anomer as an acceptor. This result indicates that it recognizes the anomeric hydroxyl group not involved directly in the reaction. ƒÀ - D-Cellobiose was also phosphorolyzed faster than the ƒ¿-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-l-phosphate for its binding site. A model of competi tive substrate inhibition was proposed, and the experimental data fit well to the theoretical values that were calculated in accordance with this model. Cellobiose phosphorylase [EC 2.4.1.20] is one of the formed during the enzymatic reaction. That of the synthetic enzymes phosphorolyzing glucosides. It catalyzes revers reaction was determined by measuring the amount of P, ible phosphorolysis of D-cellobiose into D-glucose and ƒ¿- liberated from G-1-P with an acceptor. D-glucose-1-phosphate (G-1-P) with inversion of the ano The amount of G-1-P was measured by using the phos meric configuration. It is present in Clostridium thermocel phoglucomutase-glucose-6-phosphate dehydrogenase sys lum (1), Ruminococcus flavefaciens (2), Cellvibrio gilvus tem (12). D-Glucose was measured by means of the glucose (3), Fomes annosus (4), and Cellulomonas (5, 6). Alexan oxidase-peroxidase method with mutarotase (13) using the der partially purified the enzyme from C. thermocellum (7) Glucose C Test Wako (Wako Pure Chemicals, Osaka). Pi in and synthesized several disaccharides from G-1-P and the presence of G-1-P was measured selectively by the acceptor sugars using the enzyme preparation (8). We method of Lowry and Lopez (14). reported a convenient synthetic method for D-glucosyl- Kinetic Parameters•\Kinetic parameters were calcu- D-xylose from G-1-P and D-xylose using C. gilvus cells as an ated from the experimental results following the Gauss- immobilized cellobiose phosphorylase (9). We purified the Newton method described by Cleland (15) using computer enzyme from C. gilvus to an electrophoretically homogene programs written in BASIC. ous state and reported its properties (10). We also found that its reaction proceeded through an ordered bi bi RESULTS AND DISCUSSION mechanism (11). In the present paper, an extensive kinetic study on the synthetic reaction of this enzyme is reported. Substrate Specificity of the Synthetic Reaction•\Table I indicates relative initial velocities obtained with various MATERIALS AND METHODS sugars as a glucosyl acceptor. Derivatives at C1 of the D-glucose molecule such as methyl-D-glucosides and Materials•\ƒ¿-D-Glucose-1-phosphate (G-1-P) dipotas 1,5-anhydro-D-glucitol did not serve as an acceptor. Iso sium salt, ƒÀ-D-glucose, and ƒÀ-D-cellobiose were purchased mers at C3 (D-allose), C4 (D-galactose), and C5 (L-idose) from Sigma (St. Louis, USA). ƒ¿-D-Cellobiose (containing did not serve as an acceptor. In contrast, some of the C2 about 5% of the ƒÀ-anomer) was obtained by ethanol derivatives (D-mannose, 2-deoxy-D-glucose, and D-gluco precipitation from a D-cellobiose solution. All other chemi samine) and C6 ones (6-deoxy-D-glucose and D-xylose) cals used in the experiments were of reagent grade. could act as an acceptor, although less effectively than Cellobiose phosphorylase was purified from C. gilvus cells D-glucose. These results indicate that the configurations of by the method described by Kitaoka et al. (11). the D-glucose molecule at C1, C3, C4, and C5 are strictly Assay Methods-Reactions of the cellobiose phospho required. Apart from D-glucose derivatives, ketose, pen rylase were carried out at 37•Ž in 50mM Tris-HCl buffer tose (except D-xylose), D-glucono-ƒÂ-lactone, and sugar (pH 7.0) containing 5mM MgCl, and 0.02% bovine serum alcohols had no acceptor activity. Essentially the same albumin as a stabilizer of the enzyme. One unit of the specificity for the acceptor molecule was reported with the activity was defined as the amount of the enzyme which partially purified enzyme from C. thermocellum (7). produces 1ƒÊmol of D-glucose or G-1-P per min with 10mM The acceptor specificity for the C2 derivatives is signifi D-cellobiose and 10mM inorganic phosphate (Pi) under the cantly different from that reported for a maltose phospho above conditions. The initial rate of the phosphorolysis was rylase (16). The cellobiose phosphorylase can accept an assayed by measuring the amount of G-1-P or D-glucose axial hydroxyl group at C2 (D-mannose) whereas it cannot 40 J. Biochem. Synthetic Reaction of Cellobiose Phosphorylase 41 accept bulky substitution at the C2 hydroxyl group (N-ace the case of the maltose phosphorylase, deletion (2-deoxy- tyl-D-glucosamine). On the other hand, the maltose phos D-glucose), substitution with an amino group (D-gluco phorylase cannot accept the former whereas it can accept samine) or substitution with a bulky group (N-acetyl- the latter. Furthermore, the deletion of the C2 hydroxyl D-glucosamine) did not decrease the initial rates. Cellobiose group (2-deoxy-D-glucose) or substitution with an amino phosphorylase, therefore, must have a recognition site for group (D-glucosamine) resulted in a significant decrease in the initial rate in the case of cellobiose phosphorylase. In TABLE I. Substrate specificity in the synthetic reaction. Values are indicated as ƒÊmol/min•EU. For experimental details, see the text. •\, under 0.03. TABLE 11. Apparent kinetic parameters of various substrates. Values were calculated at the following concentrations: a2-10mM, b5-100mM, c5-20mM, and d2.5-50mM. Fig. 2. The ƒÒ-[s] plots of the various substrates. •œ, D-glucose; •› , 6-deoxy-D-glucose; •£, D-mannose; •¢, 2-deoxy-D-glucose; •¡, D-glucosamine; • , D-xylose. Solid lines are calculated curves using the Michaelis-Menten equation. Fig. 1. Time course of the reactions with the substrates of both anomeric types. •›, ƒ¿-D-glucose; •œ, ƒÀ-D-glucose; •¢, ƒ¿-D-cellobiose; •£ , ƒÀ-D-cellobiose. Solid and broken lines indicate synthetic and phosphorolytic reactions, respectively. The synthetic reaction was carried out using 1mM ƒ¿- or ƒÀ-D-glucose with 10mM G-1-P, and was Fig. 3. The ƒÒ-[Glc] plot at various [G-1-P]. Initial concentra followed by measuring the amount of liberated P,. The phosphorolytic tions of G-1-P are: •£, 1mM; • , 2mM; •¡, 5mM; •›, 10mM; •œ, 20 reaction was done using 1mM ƒ¿- or ƒÀ-D-cellobiose with 10mM Pi, mM. Lines are theoretical ones. and was followed by measuring the amount of D-glucose produced. Vol. 112, No. 1, 1992 42 M. Kitaoka et al. the equatorial hydroxyl group at C2. On the other hand, (2-deoxy-D-glucose) fold, but substitution with an amino maltose phosphorylase does not appear to have this type of group increased the value only 6-fold (D-glucosamine). This recognition site, but has a structure which causes steric suggests that the amino group of D-glucosamine plays a hindrance to the axial hydroxyl group at C2. certain role in binding with the enzyme. As was already The apparent kinetic parameters for the sugars which shown in Table I, 6-deoxy-D-glucose is a good acceptor, had an acceptor activity were determined at 10mM G-1-P, after D-glucose. Table II indicates that it has a slightly as shown in Table II. These values were calculated from the higher Vmax value than that of D-glucose though its Km value experimental data using the concentration ranges indicated is about 10 times as high as that of D-glucose. Thus, the C6 in the legend, i. e. the ranges where substrate inhibition is hydroxyl group of the D-glucose molecule is thought to be negligible. It is clear that D-glucose has the lowest Km value involved only in binding of the substrate. However, loss of among the tested sugars. Compared to D-glucose, the other the hydroxymethyl group (D-xylose) resulted in a signifi five sugars have generally lower Vmaxvalues and remark- cant increase in Km and decrease in Vmax, indicating that the ably higher Km values. This indicates that the hydroxyl hydroxymethyl group is important for both binding and groups at C2 and C6 of the D-glucose molecule play more of catalysis. a role in binding of D-glucose to the active site of the enzyme Recognition of the Reducing End-Neither methyl-D- than in catalysis. Comparison of the parameters for 2- glucosides nor 1,5-anhydro-D-glucitol (1-deoxy-D-gluco deoxy- and for 6-deoxy-D-glucose revealed that the hydrox pyranose) served as an acceptor, suggesting that the yl group at C2 is much more important than that of C6. enzyme recognizes the reducing hydroxyl group of the Substitution of a proton for the C2 equatorial hydroxyl acceptor D-glucose molecule. So the reaction rates were group increased the Km values 50- (D-mannose) or 80- followed in the presence of 10mM G-1-P and 1mM of either ƒ¿- or ƒÀ-D-glucose.
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