J. Biochem. 112,,40-44 (1992)

Synthetic Reaction of Cellvibrio gilvus Cellobiose

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 from Cellvibrio gilvus were investigated in detail. It was found that, besides D-, 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 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. 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 . 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 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 , 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 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. As Fig. 1 shows, the reaction with the ƒÀ-anomer proceeded about four times faster than that with the ƒ¿-anomer. It can be concluded that the enzyme preferentially recognizes the ƒÀ-anomeric hydroxyl group of

Fig. 4. The 1/ƒÒ-1/[G-1-P] plot at various [Glc]. Initial concen trations of D-glucose are: •›, 2mM; •¢, 10mM; • , 20mM; •¤, 30mM; •ž, 40mM; •œ, 50mM; •£, 60mM; •¡, 70mM; •¥, 80mM; •Ÿ, 100mM. Fig. 5. The Kmapp- and Vmaxapp-[Glc] plots.•œ, Kmapp;••¡, Vmaxapp.

Fig. 6. A proposed model of competitive sub strate inhibition.

J. Biochem. Synthetic Reaction of Cellobiose Phosphorylase 43

D-glucose. In the phosphorolytic reaction too, the enzyme ing site of G-1-P (site 1) competitively. The fact that all of recognizes ƒÀ-D-cellobiose in preference to the a-form, as the lines were straight indicates that G-1-P does not bind to shown in Fig. 1. Usually anomeric hydroxyl groups are the binding site of D-glucose (site 2). strictly recognized by the enzymes when they are ddirectly Figure 5 shows the relationship between initial concen involved in the reactions [bond formation, oxidation (17) tration of D-glucose and apparent Vmaxand Km for G-1-P. or isomerization (18)] . However , the case described above, The Vmaxappcurve gave a typical Michaelis-Menten curve, i.e., recognition of the hydroxyl group which is not directly indicating again that the inhibition pattern of D-glucose is involved in the reaction, is quite unusual . We know of only competitive. If the inhibition were a normal competitive two instances. One is Rhizopus niveus glucoamylase report one, the obtained Kmappcurve should be straight. However, ed by Kimura and Chiba (19). Subsite 2 of this enzyme it was parabolic. This suggests strongly that some allosteric exhibited a higher affinity for ƒ¿-D-glucose than ƒÀ-D-glu effects exist in the inhibition. cose. The other was reported recently by Tsumuraya et al. Based on an ordered bi bi mechanism (11) and the (20). In this paper, they reported that a maltose phospho observation described above, a model of competitive sub rylase recognizes only ƒ¿-D-glucose as an acceptor but not ƒÀ- strate inhibition was proposed as shown in Fig. 6. In the D-glucose, and predicted that cellobiose phosphorylase model, the synthetic reaction proceeds through the follow- would act in the same way (20). The result shown in Fig. 1 ing four steps. Step 1: G-1-P binds to the enzyme at site 1. indicates that their prediction is correct. However, it is not Step 2: D-glucose binds to the enzyme-G-1-P complex at clear whether the cellobiose phosphorylase also utilizes ƒ¿- site 2. Step 3: P, is released from the enzyme-substrates D-glucose or not, because of the occurrence of mutarotation. complex. Step 4: D-cellobiose is released from the enzyme- Substrate Inhibition in the Synthetic Reaction•\The cellobiose complex. The inhibition involves the following relation between initial rate and the substrate concentra two steps. Step i-1: D-glucose binds at site 1 instead of tion of various acceptors was examined in the synthetic G-1-P. Step i-2: another D-glucose molecule binds to the reaction at a constant concentration (10mM) of G-1-P. The enzyme-glucose complex at site 2 to form a dead end results are shown in Fig. 2. As is expected, typical hyper complex. The elementary steps can be expressed in the bolic curves were obtained with acceptors such as D-man following reaction formulae. In the formulae, the phospho nose, 2-deoxy-D-glucose, and D-xylose. With these accep rolytic reaction was neglected to simplify the resulting rate tors, the reaction seems to proceed in accordance with the equation. Michaelis-Menten equation. However, some acceptors such as D-glucosamine and 6-deoxy-D-glucose gave curves differ ent from the typical hyperbolic one. This is especially prominent with D-glucose. The initial rate decreased, at D-glucose concentrations higher than 10mM, with an increase in the concentration. Similar phenomena were reported with a cellobiose phosphorylase from C. thermo cellum(7) and with a laminaribiose phosphorylase (21). This phenomenon was further studied. It is known that D-glucose acts as a competitive inhibitor against G-1-P in the cases of phosphorylase (22) and (A, G-1-P; B, D-glucose; P, Pi; Q, D-cellobiose; E, enzyme) (23), though it is not a substrate for these enzymes. In the case of the cellobiose phosphorylase, Using the steady state method, the following equations D-glucose seems to act not only as a substrate but also as a are obtained. competitive inhibitor to the other substrate, G-1-P. To v=k+3[E-AB] (6) determine whether D-glucose inhibits the binding of G-1-P competitively or not, ƒÒ-[D-glucose] curves were obtained at several concentrations of G-1-P. As shown in Fig. 3, the concentration of D-glucose giving the maximum velocity increased with an increase in the concentration of G-1-P. This result suggests that D-glucose acts as a competitive inhibitor. This inhibition can be called competitive sub strate inhibition. Chiba et al. (24) reported a similar phenomenon in a reaction of phosphoglucomutase. In this case, one of the substrates, G-1-P, competes with the other substrate, D-glucose-1,6-bisphosphate, at much higher concentrations (up to 1,000 times) than its Km value. But, in the case of the cellobiose phosphorylase, the inhibition can be observed at concentrations of D-glucose comparable [E]0=[E]+[E-A]+[E-AB]+[E-B]+[E-BB] (11) to its Km value. From the above equations, the initial rate, v, is expressed Kinetic Analysis of the Competitive Substrate Inhibi tion•\In Fig. 4, reciprocals of reaction rates are plotted as in Eq. 12. Equation 12 can be simplified as shown in Eq. against inverse concentrations of G-1-P at various concen 13 to apply the Gauss-Newton method (15). Values of a, b, c, d, e, and Vmaxwere calculated from the trations of D-glucose. The fact that the lines cross at a certain point on the vertical axis at higher D-glucose experimental data as 1.41, 1.99, 2.83, 1.43•~10-2, 3.90•~ concentrations indicates that D-glucose inhibited the bind 10-3, and 2.07 respectively. From these values, the kinetic

Vol. 112, No. 1, 1992 44 M. Kitaoka et al.

parameters were calculated as follows: Vmax=2.07ƒÊmol/ min•EU, KiA=0.5mM, KmA=2.0mM, KmB=2.8mM, KI1= REFERENCES 170mM, and KI2=3.0mM. The Vmax value of the synthetic reaction is 1.45 times larger than that of the phosphorolytic 1. Sib, C.J. & McBee, R.H. (1955) Proc. Montana Acad. Sci. 15,21- one (11). Theoretical curves obtained using these parame 22 2. Ayers, W.A. (1958) J. Bacteriol. 76, 515-517 ters fit well with plots of experimental data, as shown in 3. Hulcher, F.H. & King, K.W. (1958) J. Bacteriol. 76, 571-577 Fig. 3. This strongly supports the proposed model. 4. Huttermann, A. & Volger, C. (1973) Nature New Biol. 245, 64 In this model, the allosteric effect in inhibition can be 5. Sato, M. & Takahashi, H. (1967) Agric. Biol. Chem. 31, 470-474 explained by the presence of a cubic [B] term in the 6. Schimz, K.-L., Broll, B., & John, B. (1983) Arch. Microbiol. 135, denominator of Eq. 12. That is, Eq. 14 can be obtained from 241-249 Eq. 12. In the equation, Kmapp is expressed as the square of 7. Alexander, J.K. (1968) J. Biol. Chem. 243, 2899-2904 8. Alexander, J.K. (1968) Arch. Biochem. Biophys. 123, 240-246 [B]. This agrees with the parabolicity of the Kmapp-[B] 9. Kitaoka, M., Sasaki, T., & Taniguchi,H. (1990) Appl. Micro biol. curve in Fig. 5. Biotechnol. 34, 178-182 Likewise, Eq. 15 can be obtained from Eq. 12. 10. Sasaki, T., Tanaka, T., Nakagawa, S., & Kainuma, K. (1983) Biochem. J. 209, 803-807 11. Kitaoka, M., Sasaki, T., & Taniguchi, H. (1992) Biosci. Biotech. Biochem. 56, 652-655 This equation fits one of the Michaelis-Menten equations 12. Michael, G. (1974) in Methods of Enzymatic Analysis, 2nd and predicts the hyperbolic Vmaxapp-[B]curve shown in English ed. (Bergmeyer, H.U., ed.) Vol. 4, pp. 185-191, Aca demic Press, London and New York Fig. 5. 13. Okuda, J., Miwa, I., Maeda, K., & Tokui, K. (1977) Carbohydr. If step i-2 is omitted from the model, i.e. D-glucosedoes Res. 58, 267-270 not bind to site 2 after its binding to site 1, v can be 14. Lowry, O.H. & Lopez, J.A. (1946) J. Biol. Chem. 162, 421-428 calculated as in Eq. 16. From this equation, Kmappis 15. Cleland, W.W. (1979) in Methods in Enzymology (Purich, D., calculated as follows. ed.) Vol. 63, pp. 103-138, Academic Press, New York 16. Selinger, Z. & Schramm, M. (1961) J. Biol. Chem. 236, 2183- 2185 17. Keilin, D. & Hartree, E.F. (1952) Biochem. J. 50, 331-341 18. Schray, K.J. & Rose, I.A. (1971) Biochemistry 10, 1058-1062 19. Kimura, A. & Chiba, S. (1991) Abstracts of 13th Japanese Carbohydrate Symposium (in Japanese), pp. 127-128 It is impossible to explain the parabolicity of the observed 20. Tsumuraya, Y., Brewer, C.F., & Hehre, E.J. (1990) Arch. Kmapp-[B]curve using Eq. 17 because [B] is first-order. Biochem. Biophys. 281, 58-65 21. Goldemberg, S.H., Marechal, L.R., & De Souza, B.C. (1966) J. Thus, step i-2 must be involved in the observed inhibition. Biol. Chem. 241, 45-50 Formation of a ternary complex from one enzyme molecule 22. Helmreich, E., Michaelides, M.C., & Cori, C.F. (1967) Biochem and two D-glucose molecules is a feature of this type of istry 6, 3695-3710 competitive substrate inhibition. It can be anticipated that 23. Silverstein, R., Voet, J., Reed, D., & Abeles, R.H. (1967) J. Biol. a similar complex will be formed with maltose phospho Chem. 242, 1338-1346 rylase, laminaribiose phosphorylase, and trehalose phos 24. Chiba, H., Ueda, M., & Hirose, M. (1976) Agric. Biol. Chem. 40, 2423-2431 phorylase.

J. Biochem.