The Role of Sugar Phosphate in the Biosynthesis of Complex

19

The

R o l e of Sugar Phosphate

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Biosynthesis of

Complex

in

the

Saccharides

W. Z. HASSID Department of Biochemistry, University of California, Berkeley, Calif. 94720 Nucleoside diphosphate sugars seem to be superior donors of the glycosyl moiety for complex sugar formation because they have a higher negative free energy of hydrolysis (ΔG°) than other glycosyl compounds. This seems to be the reason why most of the polysaccharides and oligosaccharides are synthesized in vivo from these sugar nucleotides. The conditions of the reactions and the factors by which they are affected in the production of the various complex saccharides will be discussed here. Synthesis of the cell wall polysaccharides, cellulose, xylan, pectin, and otherswillalso be discussed. ' T ' h e synthesis of complex saccharides (oligosaccharides, glycosides, homo- and heteropolysaccharides) involves the process of transglycosylation. In this process the glycosyl donor may be sugar phosphate or sugar nucleotide oligosaccharide or polysaccharide. However, the most effective compounds to serve as donors for the glycosyl unit for enzymic synthesis of the complex carbohydrates have been shown to be phosphorylated sugars, especially the nucleoside diphosphate sugars. The reason for this is because they possess a high negative free energy of hydrolysis ( A G ° ' ) . The Role of Sugar Phosphate in Biosynthesis of Complex Saccharides Transglycosylation Reactions from Different Phosphate Containing Substrates. Although some oligosaccharides and polysaccharides can be synthesized in vitro from D-glucose-l-P ( J ) , it is now believed that complex saccharide formation from this phosphorylated sugar by phosphorolysis is not a normal physiological process; these enzymes act only i n a degradative capacity. Only six enzymes of this type are known: 362 Isbell; Carbohydrates in Solution Advances in Chemistry; American Chemical Society: Washington, DC, 1973.

19.

Biosynthesis of Complex Saccharides

HASSID

363

glycogen phosphorylase (glycogen muscle phosphorylase) (2), starch phosphorylase (potato phosphorylase) (3), sucrose phosphorylase (Pseudomonas sacchar&phila) (4), maltose phosphorylase (Neisseria meningitidis) ( 5 ) , cellobiose phosphorylase (Ruminococcus flavefaciens) (6), and laminaribiose phosphorylase (Euglena gracilis) (7). They are produced by the following reactions:

+

CH OH 2

H3PO4

\

CH OH

CH OH

2

•O\OH

H

CH OH

2

2

H^LAQH

BA^OH

OH

OH

H

CH OH

2

2

OH J n

H

reducing end

11

CH OH

HJ_

CH OH 2

HO H

OH

L

H

OH

H

OH

H

OH

a-D-glucopyranosyl phosphate

Formation of glycogen chains by glycogen muscle phosphorylase and starch amylose by potato phosphorylase. (Pseudomonas saccharophila) Sucrose + P i ^ a-D-glucose 1-phosphate + D-fructose. Sucrose formation by the sucrose phosphorylase reversible reaction. (Neisseria meningitidis) 4-0-a-glucosyl-D-glucose + P i ^ @-D-glucose 1-phosphate + D-glucose. (maltose) Maltose formation by the maltose phosphorylase reversible reaction. (Ruminococcus flavefaciens) 4-0-^-glucosyl-D-glucose + D-glucose se + P ii ^ a-D-glucose a-D-glucose 1-phosphate 1-phospr (cellobiose) Cellobiose formation by the cellobiose phosphorylase reversible reaction.

Isbell; Carbohydrates in Solution Advances in Chemistry; American Chemical Society: Washington, DC, 1973.

364

CARBOHYDRATES IN SOLUTION

(Euglena gracilis) 3-0-@-glucosyl-D-glucose + P i ^ a-D-glucose 1-phosphate + D-glucose. (laminaribiose) Laminaribiose formation by the laminaribiose phosphorylase reversible reaction. The number of enzymes responsible for the formation of polysaccharides from sucrose is also restricted. The two best known enzymes that form polysaccharides from sucrose are the dextran, synthesized by the microorganism Leuconostoc mesenteroides (8) and related organisms, and the levan, produced from the same substrate by Acetobacter levanicum (9) and other species. Energy Relations of the Phosphorylated Sugar Substrates. F r o m the thermodynamic point of view, nucleoside diphosphate sugars are superior donors for formation of complex saccharides because they have the highest negative free energy of hydrolysis of a l l known compounds containing glycosyl groups that can serve as a monosaccharide donor (10). Thus, the A G ° ' of uridine phosphate ( U D P ) - D - g l u c o s e at p H 7.4 is - 7 6 0 0 cal mole" while that of a-D-glucose 1-phosphate at p H 8.5 is —4800 cal mole" . The value of A G ° ' of the a-D-glucose- (1 - » 4) linkage of glycogen which is produced from these substrates is —4300 cal mole" . Since the A G ° ' of hydrolysis of UDP-D-glusose is —7600, the free energy change during the formation of glycogen from UDP-D-glucose can be calculated as —3300 mole" . This value corresponds to an equilibrium of about 250, which amounts to a practically quantitative conversion of the nucleotide bound glucose into glycogen. 1

1

1

1

Glycosyl—Enzyme Complex Intermediates in Biosynthesis of Complex Saccharides. The synthesis of nucleoside diphosphate sugars involves the transfer of a nucleotidyl group from a nucleoside triphosphate to a sugar 1-phosphate with the simultaneous release of pyrophosphate according to the following general reaction ( I I ) : Nucleoside triphosphate + sugar 1-phosphate J\

pyrophosphorylase

nucleoside diphosphate sugar +

pyrophosphate

M a n y nucleoside diphosphate sugars containing different bases and different sugar moieties were found to be synthesized by this enzymic process. A similar reaction was observed (12) that leads to the synthesis of a nucleoside monophosphate sugar which occurs as follows: cytidine triphosphate + iV-acetylneuraminic acid —* cytidine monophosphate A -acetylneuraminic acid + pyrophosphate. r


19.

HASSID


365

As previously mentioned, sucrose can be formed from a-r>glucose 1-P and D-fructose by a sucrose phosphorylase reversible reaction: Sucrose + P i ^ a-D-glucose-l-P + D-fructose. The glucosyl phosphate i n this reaction does not seem to be an essential product or substrate of sucrose phosphorylase activity for the synthesis of disaccharides. This ester can be regarded as one of several glucose donors for the enzyme. The sucrose phosphorylase can act not only as a phosphorylase but also as a transglucosylase capable of mediating the transfer of the D-glucose portion of substrate to a variety of acceptors (1). The evidence for the double function of the enzyme is cited from the observation that when P-labeled inorganic phosphate and nonradioactive a-D-glucose 1-P are added to sucrose phosphorylase preparations in the absence of ketose sugars, a rapid redistribution of the isotope occurs between the organic and inorganic fractions without the liberation of D-glucose. This observation led to the assumption that the enzyme combines reversibly with the D-glucose of a-D-glucose 1-P, forming a D-glucose— enzyme complex and releasing inorganic phosphate, according to the equation: 32

a-D-glucose-l-P + enzyme ^ D-glucose-enzyme + P i The equilibrium reaction would require that the energy of the a-Dglucose-l-P linkage be preserved i n the D-glucose-enzyme bond. The transfer of phosphate could not involve the formation of free D-glucose because if this occurred, about 4800 cal mole" would be released i n the decomposition of the ester and would be required for its resynthesis. Since no external source of energy would be available for the resynthesis of the ester, it can be concluded that the original bond energy is conserved i n the D-glucose-enzyme complex. 1

The enzyme is really a glucosyl transfer agent, as shown by the catalysis of an exchange of glycosidic bonds i n the absence of phosphate (13): ^-D-fructofuranosyl a-D-glucopyranoside + L-sorbose ^ (sucrose) D-glucopyranosyl L-sorboside + D-fructose. In a similar manner, sucrose can be prepared by a reaction between D-fructose and the corresponding disaccharide containing D-threo-pentulose (14). Supporting evidence for the formation of a D-glucose-enzyme complex i n reactions catalyzed by sucrose phosphorylase from Pseudomonas


366


saccharophila was obtained by Voet and Abeles (15). They showed that when sucrose phosphorylase is denatured after exposure to uniformly labeled sucrose- C , the denatured protein contains firmly bound D-glucose or a compound derived from the D-glucose moiety of sucrose. The D-fructose moiety of sucrose is not bound to the protein. The molecular weight of the enzyme was shown (16) to be 80,000-100,000. Recently, the formation of a covalent glycosyl-enzyme intermediate was also shown by Bell and Koshland (17) i n another reaction. Evidence was presented that the mechanism of the enzyme, phosphoribosyl-adenosine triphosphate: pyrophosphate phosphoribosyl transferase, proceeds through a covalent phosphoribosyl-enzyme intermediate. The intermediate has been demonstrated after incubating the enzyme with C - 5 phosphoribosyl-l-pyrophosphate ( P R P P ) under native and denaturing conditions. The intermediate also forms from the reverse direction as shown when the enzyme is mixed with its product N-(5-phosphoribosyladenosine triphosphate ( P R - A T P ) . These data give evidence for a covalent enzyme-substrate intermediate. The enzyme which catalyzes the overall reaction proceeds as follows: NH (g)-CH r-jsk^N 1 4

14

2

2

2

(£H|He)-CH

PRPP

+

ATP

(D-CH

®-®-(P)-CH

PR-ATP

+

PPi

Two glycosyl-enzyme intermediates have thus far been definitely shown to be formed, and only one involved i n the formation of a disaccharide (sucrose) is known. However, it is now believed that a l l oligo-


19.

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HASSID

saccharides and polysaccharides are synthesized by transglycosylation via glycosyl-enzyme intermediates. A search of the literature by Bell and Koshland (18) showed that during the last decade 58 cases have been recorded for which there is strong evidence for covalent enzyme-substrate intermediates involving various enzymes other than transglycosylases; these include phosphoryl-, acyl-, acetyl-enzyme, and other intermediates. Enzymic transfer of a glycosyl moiety from a donor substrate to an acceptor may result i n a product with the same configuration of the anomeric carbon atom as the original substrate or i n an inversion. Natural complex saccharides found to be formed from a particular substrate with an inversion of configuration seem to be more prevalent than those without inversion. Koshland (19) has postulated a theory of transglycosylation that accounts for both types:

H

0/R

G

En-

+ UDP:

G

\H

4) glucosyl linkages. It has also been shown that mung beans, peas, and other plants contain a pyrophosphorylase which forms GDP-D-glucose from a-D-glucose 1-P and G T P . Based on the data obtained with enzymic plant preparations, we proposed the following mechanism for cellulose synthesis: 14

Guanosine triphosphate + a-D-glucose 1-phosphate pyrophosphorylase guanosine diphosphate D-glucose + pyrophosphate transferase n (Guanosine diphosphate D-glucose) + acceptor > acceptor-(@-l,4-D-glucose) + n(guanosine diphosphate) (cellulose) n

However, other workers claim that UDP-D-glucose may also be an effective donor for cellulose formation with preparations from higher plants, but we could not substantiate their results i n our laboratory. Subsequently, Ordin and H a l l (25,26) found that particulate preparations from oat coleoptiles could use UDP-D-glucose as substrate for polysaccharide formation. U p o n degradation of the polysaccharide derived from UDP-D-glucose w i t h impure cellulase, cellobiose, and to a lesser extent a substance identified as a trisaccharide containing mixed / ? - ( l -> 4 ) , / ? - ( l —> 3) glucosyl linkages were obtained. These results with enzyme preparations from oat coleoptiles and UDP-D-glucose as substrate could be substantiated by chemical methods (27). However, the question remained whether a single enzyme is i n volved i n the synthesis of this oat coleoptile polysaccharide containing mixed linkages or whether two enzymes are present, each forming one of the two linkages of this polymer(s)—i.e., one synthesizing / ? - ( l - » 4 ) and the other the / ? - ( l -> 3) linkage. Concerning this problem, we have observed that when a 1 X 10" M UDP-D-glucose substrate concentration with particulate or digitonin solubilized enzyme preparations from oat coleoptiles was used, a / ? - ( l - » 3 ) glucan is formed as the main product. Glucan produced from a U D P - D glucose of 1 X 10" M or lower concentration contained practically only / ? - ( l - » 4 ) glucosyl linkages. Also, a separation of the / ? - ( l - > 4 ) and / ? - ( l - » 3 ) glucan synthetase activities could be achieved at 1 X 10" M UDP-D-glucose concentration when the digitonin solubilized enzyme was adsorbed on a hydroxylapatite gel and then eluted with strong potassium phosphate buffer (28). 3

5

3

The results indicate that the particulate enzyme contains two enzymes; using UDP-D-glucose as substrate, one is capable of synthesizing


370


£ - ( l - * 4 ) and another / ? - ( l - » 3 ) linkages. The Km of the 0 - ( l - > 4 ) synthesizing enzyme was found to be 1.2 X 10" M and that of the 0 - ( l - > 3 ) 6 X 1 0 " M (29). Cellulose is the main component of the cell-wall which forms the insoluble skeletal framework of all higher plants. This polymer is associated with other polysaccharides, chiefly xylan, pectin, glucomannan, and hemicelluloses. These polysaccharides have been synthesized in vitro from various sugar nucleotides with enzymes isolated from plant sources. Xylan. Experiments in vivo by several investigators (30) indicated that xylan i n plants originated from glucose by a series of reactions first leading to the formation of pentose polysaccharide. UDP-D-xylose has been isolated from plant seedlings and synthesized enzymically from UDP-D-glucose by the following sequence of reactions: 5

4

UDP-D-glucose

dehydrogenase > UDP-D-glucuronic acid decarboxylase UDP-D-xylose

Particulate preparations from corn shoots readily incorporate r e labeled D-xylose from UDP-D-xylose- C into a polysaccharide i n which the D-xylose residues are combined by /?-l,4-D-xylosyl bonds (31). It was shown that this polysaccharide, similar to natural plant xylan, contains a small proportion of L-arabinose units which have the furanose configuration. Pectins. The basic building unit of pectins is known to be «-l,4 linked D-galacturonic acid which forms the polygalacturonic acid chain. The carboxyl groups of the D-galacturonic acid i n the chain are methylated to various degrees. It has been shown that UDP-D-galacturonic acid is present in higher plants (32) and that they contain enzymes which lead to the formation of this uronic acid nucleotide, starting with UDP-D-glucose by the following pathway (33,34): 14

UDP-D-Glucose

dehydrogenase epimerase > UDP-D-glucuronic acid — UDP-D-galacturonic acid.

UDP-D-galacturonic acid has also been isolated from mung beans. A particulate preparation from mung beans was found to catalyze the polymerization of the D-galacturonic acid from the UDP-D-galacturonic acid, resulting i n the formation of a polygalacturonic acid chain (35). The synthetic polygalacturonate could be hydrolyzed with Penicillum chrys-


19.

371


HASSID

ogenum polygalacturonase to D-galacturonic acid and with an exo-polygalacturonic acid transeliminase from Clostridium multifermentans to unsaturated 4,5-digalacturonic acid. The action of these enzymes is specific for degradation of the polygalacturonic acid chain (36,37). The structure of the unsaturated digalacturonic acid was shown to be as follows (38): COOH

H

OH

H

Kauss (39) has shown that the esterification of the carboxyl groups in the D-galacturonic acid chain takes place by a transfer of the methyl groups from S-adenosyl-L-methionine, analogous to the case i n which the 4-methyl ether groups are transferred to D-glucuronic acid of hemicellulose (40). Glucomannan. Elbein (41, 42) has shown that when GDP-D-mannose- C is used as substrate, a radioactive glucomannan is synthesized and that M g is required for its formation. The addition of unlabeled GDP-D-glucose to the reaction mixture containing GDP-D-mannose- C resulted i n a marked inhibition of incorporation of radioactive D-mannose into an insoluble polysaccharide. The enzyme(s) involved i n the synthesis of glucomannan apparently has a greater affinity for GDP-D-glucose than for GDP-D-mannose, which would account for the inhibition of mannose incorporation by GDP-D-glucose. Several of the oligosaccharides obtained from degradation of the glucomannan contained D-glucose and D-mannose i n various proportions, indicating that the glucomannan is not a mixture of cellulose and mannan. The D-mannose units i n this polysaccharide were shown to be linked by £-1,4 bonds. 14

2 +

14

The fact that glucomannan is obtained from GDP-D-mannose as substrate suggests that the particulate enzyme contains an epimerase which converts GDP-D-mannose to GDP-D-glucose. However, such an epimerase has not been observed. In this connection cellulose isolated from wood contains a considerable amount of D-mannose. Hemicellulose. The hemicelluloses consisting of hexoses, pentoses, and uronic acids may be separated into two fractions, A and B. H e m i cellulose B usually contains a higher proportion of uronic acid, mainly 4-methyl-D-glucuronic acid, than the A fraction. This methyl derivative of D-glucuronic acid is most frequently isolated i n combined form as the


372


4-methyl aldobiuronic acid because it is hydrolyzed with difficulty with acid. As previously mentioned, Kauss (40) has shown that the methyl donor for the formation of 4-methyl-D-glucuronic acid of hemicellulose B proved to be S-adenosyl-L-methionine, the same as i n pectin. A particulate preparation from immature corn cobs containing hemicellulose B was found capable of transferring the C-labeled methyl group from S-adenosyl-L-methionine to a macromolecular acceptor present i n the particles. The radioactive product was shown to be hemicellulose B labeled i n the 4-methyl-D-glucuronic acid residues. It was isolated chiefly as 4-methylglucuronosyl-( 1 —» 2)-D-xylose. In another experiment with the particulate enzyme from the same plant he obtained 4-methyl D-glucuronosyl-D-galactose together with 4-methyl D-glucuronosyl-D-xylose. Kauss (43) also found that the particulate enzyme preparation from immature corn cobs contains, i n addition to the methyl transferase, an enzyme which introduces the D-glucuronic acid group from UDP-D-glucuronic acid into hemicellulose B. The observation that the methyl ether or ester groups of plant heteropolysaccharides are introduced at the macromolecular level is similar to the finding that C - and N-methyl groups of R N A and D N A are also introduced into preformed macromolecules. While we are far from knowing a l l the biochemical and physiological details of the synthesis of these polymers, we obtained information pertaining to basic biochemical reactions involved i n their synthesis. 14

Literature Cited 1. Hassid, W. F., "The Carbohydrates, Chemistry and Biochemistry," p. 301, Vol. IIA, W. Pigman and D. Horton, Eds., 2nd ed., Academic, 1970. 2. Cori, G. T., Cori, C. F., J. Biol Chem. (1940) 135, 733. 3. Hanes, C. S., Proc. Roy. Soc. (London) B128, 421; ibid. (1941) B129, 174. 4. Hassid, W. Z., Doudoroff, M., Barker, H. A., J. Amer. Chem. Soc. (1944) 66, 1416. 5. Fitting, C., Doudoroff, M., J. Biol. Chem. (1952) 199, 153. 6. Ayers, W. A., J. Biol. Chem. (1959) 234, 2819. 7. Marechal, L. R., Goldenberg, S. H., Biochem. Biophys. Res. Commun. (1963) 13, 106. 8. Stacey, M., Barker, S. A., "Polysaccharides of Microorganisms," p. 136, Oxford University, London and New York, 1960. 9. Hestrin, S., Feingold, D. S., Avigad, G., Biochem. J. (1956) 64, 340. 10. Leloir, C. F., Cardini, C. E., Cabib, E., "Comparative Biochemistry," p. 97, M. Florkin and H. S. Mason, Eds., Vol. 2, Academic, New York, 1960. 11. Munch-Peterson, A., Kalckar, H. M., Culoto, E., Smith, E. E. B., Nature (1953) 172, 1037. 12. Roseman, S., Proc.Natl.Acad. Sci. U. S. (1962) 48, 437. 13. Hassid, W. Z., Doudoroff, M., Advan. Enzymol. (1950) 10, 123. 14. Kalckar, H. M., "The Mechanism of Enzyme Action," p. 675, W. D. Mc Elroy and G. Glass, Eds., Johns Hopkins, Baltimore, 1953. 15. Voet, J., Abeles, R. H., J. Biol. Chem. (1967) 242, 1338.


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16. Silverstein, R., Voet, J., Reed, D., Abeles, R. H., J. Biol Chem. (1967) 242. 1338. 17. Bell, R. M., Koshland, D. E., Jr., Biochem. Biophys. Res. Commun. (1970) 38, 539. 18. Bell, R. M., Koshland, D. E., Jr., Science (1971) 172, 1253. 19. Koshland, D. E., Jr., "Symposium on the Mechanism of Enzyme Action," p. 608, W. D. McElroy, and B. Glen, Eds., Johns Hopkins, Baltimore, 1954. 20. Elbein, A. D., Barber, G. A., Hassid, W. Z., J. Amer. Chem. Soc. (1964) 86, 309. 21. Barber, G. A., Elbein, A. D., Hassid, W. Z., J. Biol. Chem. (1964 ) 239, 4056. 22. Babad, H., Hassid, W. Z., J. Biol. Chem. (1966) 241, 2672. 23. Leloir, L.F., Cardini, C. A., J. Biol. Chem. (1957) 79, 6340. 24. Lin, T. S., Hassid, W. Z., J. Biol. Chem. (1966) 241, 3282, 5284. 25. Ordin, L., Hall, M. A., Plant Physiol. (1967) 42, 205. 26. Ordin, L., Hall, M. A., Plant Physiol. (1968) 43, 473. 27. Flowers, N. H., Batra, K. K , Kemp, J., Hassid, W. Z., Plant Physiol (1968) 43 1703. 28. Tsai, C. M., Hassid, W. Z., Plant Physiol (1971) 47, 740. 29. Tsai, C. M., Hassid, W. Z., in preparation. 30. Hassid, W. Z., Neufeld, E. F., Feingold, D. S., Proc. Natl. Acad. Sci. U. S. (1959) 45, 905. 31. Bailey, R. W., Hassid, W. Z., Proc. Natl Acad. Sci. U. S., 56, 1586. 32. Neufeld, E. F., Feingold, D. S., Biochem. Biophys. Acta (1961) 53, 589. 33. Strominger, J. L., Mapson, L. W., Biochem. J. (1957) 66, 567. 34. Feingold, D. S., Neufeld, E. F., Hassid, W. Z., J. Biol. Chem. (1960) 235, 910. 35. Villemez, C. L., Lin, T. S., Hassid, W. Z., Proc.Natl.Acad. Sci. U. S. (1965) 54, 1626. 36. Macmillan, J. D., Vaughn, R. H., Biochemistry (1964) 3, 564. 37. Macmillan, J. D., Phaff, H. J., Vaughn, R. H., Biochemistry (1964 ) 3, 564. 38. Hasegawa, S., Nagel, C. W., J. Biol. Chem. (1962) 237, 619. 39. Kauss, H., Hassid, W. Z., J. Biol. Chem. (1967) 242, 3449. 40. Kauss, H., Hassid, W. Z., J. Biol. Chem. (1967) 242, 1680. 41. Elbein, A., Hassid, W. Z., Biochem. Biophys. Res. Commun. (1966) 23, 311. 42. Elbein, A. D., J. Biol. Chem. (1969) 244, 1608. 43. Kauss,H.,Biochim. Biophys. Acta (1967) 148, 572. RECEIVED December 2, 1971.


The Role of Sugar Phosphate in the Biosynthesis of Complex

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