Probing the catalytic site of rabbit muscle glycogen phosphorylase using a series of specifically modified maltohexaose derivatives


Glycogen phosphorylase (GP) is an allosteric enzyme whose catalytic site comprises six subsites (SG1, SG−1, SG−2, SG−3, SG−4, and SP) that are complementary to tandem five glucose residues and one inorganic phosphate molecule, respectively. In the catalysis of GP, the nonreducing-end glucose (Glc) of the maltooligosaccharide substrate binds to SG1 and is then phosphorolyzed to yield glucose 1-phosphate. In this study, we probed the catalytic site of rabbit muscle GP using pyridylaminated-maltohexaose (Glcα1–4Glcα1–4Glcα1–4Glcα1–4Glcα1–4GlcPA, where GlcPA = 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol]; abbreviated as PA-0) and a series of specifically modified PA-0 derivatives (Glc m -AltNAc-Glc n -GlcPA, where m + n = 4 and AltNAc is 3-acetoamido-3-deoxy-D-altrose). PA-0 served as an efficient substrate for GP, whereas the other PA-0 derivatives were not as good as the PA-0, indicating that substrate recognition by all the SG1 SG−4 subsites was important for the catalysis of GP. By comparing the initial reaction rate toward the PA-0 derivatives (V derivative) with that toward PA-0 (V PA-0), we found that the value of V derivative/V PA-0 decreased significantly as the level of allosteric activation of GP increased. These results suggest that some conformational changes have taken place in the maltooligosaccharide-binding region of the GP catalytic site during allosteric regulation.

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Fig. 5









2,5-dihydroxybenzoic acid


Glycogen debranching enzyme




α-D-glucose 1-phosphate




Glycogen phosphorylase


High-performance liquid chromatography


Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry


Molecular weight





Pi :

Inorganic phosphate


  1. 1.

    Sillerud, L.O., Shulman, R.G.: Structure and metabolism of mammalian liver glycogen monitored by carbon-13 nuclear magnetic resonance. Biochemistry. 22, 1087–1094 (1983)

    CAS  Article  PubMed  Google Scholar 

  2. 2.

    Matsui, M., Kakuta, M., Misaki, A.: Comparison of the unit-chain distributions of glycogens from different biological sources, revealed by anion exchange chromatography. Biosci Biotechnol Biochem. 57, 623–627 (1993)

    CAS  Article  Google Scholar 

  3. 3.

    Roach, P.J., Depaoli-Roach, A.J., Hurley, T.D., Tagliabracci, V.S.: Glycogen and its metabolism: some new developments and old themes. Biochem J. 441, 763–787 (2012)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Titani, K., Koide, A., Hermann, J., Ericsson, L.H., Kumar, S., Wade, R.D., Walsh, K.A., Neurath, H., Fisher, E.H.: Complete amino acid sequence of rabbit muscle glycogen phosphorylase. Proc Natl Acad Sci U S A. 74, 4762–4766 (1977)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Tagaya, M., Fukui, T.: Catalytic reaction of glycogen phosphorylase reconstituted with a coenzyme-substrate conjugate. J Biol Chem. 259, 4860–4865 (1984)

    CAS  PubMed  Google Scholar 

  6. 6.

    Gordon, R.B., Brown, D.H., Brown, B.I.: Preparation and properties of the glycogen-debranching enzyme from rabbit liver. Biochim Biophys Acta. 289, 97–107 (1972)

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Nakayama, A., Yamamoto, K., Tabata, S.: Identification of the catalytic residues of bifunctional glycogen debranching enzyme. J Biol Chem. 276, 28824–28828 (2001)

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Sato, S., Ohi, T., Nishino, I., Sugie, H.: Confirmation of the efficiency of vitamin B6 supplementation for McArdle disease by follow-up muscle biopsy. Muscle Nerve. 45, 436–440 (2012)

    Article  PubMed  Google Scholar 

  9. 9.

    Voet, D., Voet, J.D.: Biochemistry (third edition) pp. 626–656. John Wiley & sons Inc. Hoboken. (2004)

  10. 10.

    Berg, J.M., Tymoczko, J.L., Stryer, L.: Biochemistry (sixth edition) pp. 592–616. W. H. Freeman and company. N Y. (2007)

  11. 11.

    Miyagawa, D., Makino, Y., Sato, M.: Sensitive, nonradioactive assay of phosphorylase kinase through measurement of enhanced phosphorylase activity towards fluorogenic dextrin. J Biochem. 159, 239–246 (2016)

    CAS  Article  PubMed  Google Scholar 

  12. 12.

    Madsen, N.B., Shechosky, S., Fletterick, R.J.: Site-site interactions in glycogen phosphorylase b probed by ligands specific for each site. Biochemistry. 22, 4460–4465 (1983)

    CAS  Article  PubMed  Google Scholar 

  13. 13.

    Lowry, O.H., Schult, D.W., Passonneau, J.V.: Effects of adenylic acid on the kinetics of muscle phosphorylase a. J Biol Chem. 239, 1947–1953 (1964)

    CAS  PubMed  Google Scholar 

  14. 14.

    Rush, J.W.E., Spriet, L.L.: Skeletal muscle glycogen phosphorylase a kinetics: effects of adenine nucleotides and caffeine. J Appl Physiol. 91, 2071–2078 (2001)

    CAS  PubMed  Google Scholar 

  15. 15.

    Tanabe, S., Kobayashi, M., Matsuda, K.: Yeast glycogen phosphorylase: kinetic properties compared with muscle and potato enzymes. Agric Biol Chem. 52, 757–764 (1988)

    CAS  Google Scholar 

  16. 16.

    Kasvinsky, P.J., Madsen, N.B., Fletterick, R.J., Sygusch, J.: X-ray crystallographic and kinetic studies of oligosaccharide binding to phosphorylase. J Biol Chem. 253, 1290–1296 (1978)

    CAS  PubMed  Google Scholar 

  17. 17.

    Sprang, S.R., Goldsmith, E.J., Fletterick, R.J., Withers, S.G., Madsen, N.B.: Catalytic site of glycogen phosphorylase: structure of the T state and specificity for α-D-glucose. Biochemistry. 21, 5364–5371 (1982)

    CAS  Article  PubMed  Google Scholar 

  18. 18.

    Withers, S.G., Madsen, N.B., Sprang, S.R., Fletterick, R.J.: Catalytic site of glycogen phosphorylase: structural changes during activation and mechanistic implications. Biochemistry. 21, 5372–5382 (1982)

    CAS  Article  PubMed  Google Scholar 

  19. 19.

    Hiromi, K.: Interpretation of dependency of rate parameters on the degree of polymerization of substrate in enzyme-catalyzed reactions. Evaluation of subsite affinities of exo-enzyme. Biochem Biophys Res Commun. 40, 1–6 (1970)

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Hiromi, K., Nitta, Y., Numata, C., Ono, S.: Subsite affinities of glucoamylase: examination of the validity of the subsite theory. Biochim Biophys Acta. 302, 362–375 (1973)

    CAS  Article  PubMed  Google Scholar 

  21. 21.

    Nitta, Y., Mizushima, M., Hiromi, K., Ono, S.: Influence of molecular structures of substrates and analogues on taka-amylase a catalyzed hydrolyses. I Effect of chain length of linear substrates J Biochem. 69, 567–576 (1971)

    CAS  PubMed  Google Scholar 

  22. 22.

    Konishi, Y., Kitazato, S., Nakatani, N.: Partial purification and characterization of acid and neutral α-glucosidases from preclimacteric banana pulp tissues. Biosci Biotechnol Biochem. 56, 2046–2051 (1992)

    CAS  Article  Google Scholar 

  23. 23.

    Fujita, K., Tahara, T., Koga, T., Imoto, T.: Enzymatic synthesis of specifically modified linear oligosaccharides from γ-cyclodextrin derivatives. Study on importance of acive sites of Taka amylase A. Bull Chem Soc Jpn. 62, 3150–5154 (1989)

  24. 24.

    Croft, A.P., Bartsch, R.A.: Synthesis of chemically modified cyclodextrins. Tetrahedron. 39, 1417–1474 (1983)

    CAS  Article  Google Scholar 

  25. 25.

    Walker, G.J., Whelan, W.J.: The mechanism of carbohydrase action: 8, structures of the muscle-phosphorylase limit dextrins of glycogen and amylopectin. Biochem J. 76, 264–268 (1960)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Hase, S., Ikenaka, T., Matsushima, Y.: Structure analyses of oligosaccharides by tagging of the reducing end sugars with a fluorescent compound. Biochem Biophys Res Commun. 85, 257–263 (1978)

    CAS  Article  PubMed  Google Scholar 

  27. 27.

    Kuraya, N., Hase, S.: Release of O-linked sugar chains from glycoproteins with anhydrous hydrazine and pyridylamination of the sugar chains with improved reaction conditions. J Biochem. 112, 122–126 (1992)

    CAS  Article  PubMed  Google Scholar 

  28. 28.

    Makino, Y., Omichi, K.: Acceptor specificity of 4-α-glucanotransferases of mammalian glycogen debranching enzymes. J Biochem. 139, 535–541 (2006)

    CAS  Article  PubMed  Google Scholar 

  29. 29.

    Natsuka, S., Masuda, M., Sumiyoshi, W., Nakakita, S.: Improved method for drawing of a glycan map, and the first page of glycan atlas, which is a compilation of glycan maps for a whole organism. PLoS One. 9, e102219 (2014)

    Article  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Makino, Y., Omichi, K., Hase, S.: Analysis of oligosaccharide structures from the reducing end terminal by combining partial acid hydrolysis and a two-dimensional sugar map. Anal Biochem. 264, 172–179 (1998)

    CAS  Article  PubMed  Google Scholar 

  31. 31.

    Day, A.G., Parsonage, D., Ebel, S., Brown, T., Fersht, A.R.: Barnase has subsites that give rise to large rate enhancements. Biochemistry. 31, 6390–6395 (1992)

    CAS  Article  PubMed  Google Scholar 

  32. 32.

    Buckle, A.M., Fersht, A.R.: Subsite binding in an RNase: structure of a barnase–tetranucleotide complex at 1.76-Å resolution. Biochemistry. 33, 1644–1653 (1994)

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Burkhardt, G., Wegener, G.: Glycogen phosphorylase from flight muscle of the hawk moth Manduca sexta: purification and properties of three interconvertible forms and the effect of flight on their interconversion. J Comp Physiol B. 164, 261–271 (1994)

    CAS  Article  Google Scholar 

  34. 34.

    Makino, Y., Omichi, K.: Sensitive assay of glycogen phosphorylase activity by analysing the chain-lengthening action on a fluorogenic maltooligosaccharide derivative. J Biochem. 146, 71–76 (2009)

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Lineweaver, H., Burk, D.: The determination of enzyme dissociation constants. J Am Chem Soc. 56, 658–666 (1934)

    CAS  Article  Google Scholar 

  36. 36.

    Makino, Y., Fujii, Y., Taniguchi, M.: Properties and functions of the storage sites of glycogen phosphorylase. J Biochem. 157, 451–458 (2015)

    CAS  Article  PubMed  Google Scholar 

  37. 37.

    Barford, D., Johnson, L.N.: The allosteric transition of glycogen phosphorylase. Nature. 340, 609–616 (1989)

    CAS  Article  PubMed  Google Scholar 

  38. 38.

    Buchbinder, J.L., Rath, V.L., Fletterick, R.J.: Structural relationships among regulated and unregulated phosphorylases. Annu Rev Biophys Biomol Struct. 30, 191–209 (2001)

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Leonidas, D.D., Oikonomakos, N.G., Papageorgiou, A.C., Xenakis, A., Cazianis, C.T., Bem, F.: The ammonium sulfate activation of phosphorylase b. FEBS Lett. 261, 23–27 (1990)

    CAS  Article  PubMed  Google Scholar 

  40. 40.

    Ishimizu, T., Hase, S.: Substrate recognition by sugar chain-related enzymes: recognition of a large area of substrates and its strictness and tolerance. Trends Glycosci Glycotechnol. 17, 215–227 (2005)

    CAS  Article  Google Scholar 

  41. 41.

    Okubo, M., Horinishi, A., Takeuchi, M., Suzuki, Y., Sakura, N., Hasegawa, Y., Igarashi, T., Goto, K., Tahara, H., Uchimoto, S., Omichi, K., Kanno, H., Hayasaka, K., Murase, T.: Heterogeneous mutations in the glycogen-debranching enzyme gene are responsible for glycogen storage disease type IIIa in Japan. Hum Genet. 106, 108–115 (2000)

    CAS  Article  PubMed  Google Scholar 

  42. 42.

    Zhai, L., Feng, L., Xia, L., Yin, H., Xiang, S.: Crystal structure of glycogen debranching enzyme and insights into its catalysis and disease-causing mutations. Nat Commun. 7, 11229 (2016)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. 43.

    Watanabe, Y., Makino, Y., Omichi, K.: Donor substrate specificity of 4-α-glucanotransferase of porcine liver glycogen debranching enzyme and complementary action to glycogen phosphorylase on debranching. J Biochem. 143, 435–440 (2008)

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Somsak, L., Czifrak, K., Toth, M., Bokor, E., Chrysina, E.D., Alexacou, K.M., Hayes, J.M., Tiraidis, C., Lazoura, E., Leonidas, D.D., Zographos, S.E., Oikonomakos, N.: New inhibitors of glycogen phosphorylase as potential antidiabetic agents. Curr Med Chem. 15, 2933–2983 (2008)

    CAS  Article  PubMed  Google Scholar 

  45. 45.

    Martin, W.H., Hoover, D.J., Armento, S.J., Stock, I.A., McPherson, R.K., Danley, D.E., Stevenson, R.W., Barrett, E.J., Treadway, J.L.: Discovery of a human liver glycogen phosphorylase inhibitor that lowers blood glucose in vivo. Proc Natl Acad Sci U S A. 95, 1776–1781 (1998)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Lerin, C., Montell, E., Nolasco, T., Garcia-Rocha, M., Guinovart, J.J., Gomez-Foix, A.M.: Regulation of glycogen metabolism in cultured human muscles by the glycogen phosphorylase inhibitor CP-91149. Biochem J. 378, 1073–1077 (2004)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Oikonomakos, N.G., Chrysina, E.D., Kosmopoulou, M.N., Leonidas, D.D.: Crystal structure of rabbit muscle glycogen phosphorylase a in a complex with a potential hypoglycaemic drug at 2.0 Å resolution. Biochim Biophys Acta. 1647, 325–332 (2003)

    CAS  Article  PubMed  Google Scholar 

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Correspondence to Yasushi Makino.

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Nakamura, M., Makino, Y., Takagi, C. et al. Probing the catalytic site of rabbit muscle glycogen phosphorylase using a series of specifically modified maltohexaose derivatives. Glycoconj J 34, 563–574 (2017).

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  • Glycogen
  • Glycogen phosphorylase
  • Modified maltooligosaccharide
  • Pyridylamination
  • Substrate recognition