Intersubunit communication in glycogen phosphorylase influences substrate recognition at the catalytic sites
Abstract Glycogen phosphorylase (GP) is biologically active as a dimer of identical subunits, each activated by phosphorylation of the serine-14 residue. GP exists in three interconvertible forms, namely GPa (di-phosphorylated form), GPab (mono-phosphorylated form), and GPb (non-phosphorylated form)...
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| Format: | Article |
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Springer
2024-02-01
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| Series: | Amino Acids |
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| Online Access: | https://doi.org/10.1007/s00726-023-03362-6 |
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| author | Nahori Kamada Ayato Ikeda Yasushi Makino Hiroshi Matsubara |
| author_facet | Nahori Kamada Ayato Ikeda Yasushi Makino Hiroshi Matsubara |
| author_sort | Nahori Kamada |
| collection | DOAJ |
| description | Abstract Glycogen phosphorylase (GP) is biologically active as a dimer of identical subunits, each activated by phosphorylation of the serine-14 residue. GP exists in three interconvertible forms, namely GPa (di-phosphorylated form), GPab (mono-phosphorylated form), and GPb (non-phosphorylated form); however, information on GPab remains scarce. Given the prevailing view that the two GP subunits collaboratively determine their catalytic characteristics, it is essential to conduct GPab characterization to gain a comprehensive understanding of glycogenolysis regulation. Thus, in the present study, we prepared rabbit muscle GPab from GPb, using phosphorylase kinase as the catalyst, and identified it using a nonradioactive phosphate-affinity gel electrophoresis method. Compared with the half-half GPa/GPb mixture, the as-prepared GPab showed a unique AMP-binding affinity. To further investigate the intersubunit communication in GP, its catalytic site was probed using pyridylaminated-maltohexaose (a maltooligosaccharide-based substrate comprising the essential dextrin structure for GP; abbreviated as PA-0) and a series of specifically modified PA-0 derivatives (substrate analogs lacking part of the essential dextrin structure). By comparing the initial reaction rates toward the PA-0 derivative (V derivative) and PA-0 (V PA-0), we demonstrated that the V derivative/V PA-0 ratio for GPab was significantly different from that for the half-half GPa/GPb mixture. This result indicates that the interaction between the two GP subunits significantly influences substrate recognition at the catalytic sites, thereby providing GPab its unique substrate recognition profile. |
| format | Article |
| id | doaj-art-2ffacb5f14d34703b3464f92fd918d59 |
| institution | OA Journals |
| issn | 1438-2199 |
| language | English |
| publishDate | 2024-02-01 |
| publisher | Springer |
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| series | Amino Acids |
| spelling | doaj-art-2ffacb5f14d34703b3464f92fd918d592025-08-20T01:57:16ZengSpringerAmino Acids1438-21992024-02-015611910.1007/s00726-023-03362-6Intersubunit communication in glycogen phosphorylase influences substrate recognition at the catalytic sitesNahori Kamada0Ayato Ikeda1Yasushi Makino2Hiroshi Matsubara3Department of Chemistry, Graduate School of Science, Osaka Prefecture UniversityDepartment of Chemistry, Graduate School of Science, Osaka Prefecture UniversityDepartment of Chemistry, Graduate School of Science, Osaka Prefecture UniversityDepartment of Chemistry, Graduate School of Science, Osaka Prefecture UniversityAbstract Glycogen phosphorylase (GP) is biologically active as a dimer of identical subunits, each activated by phosphorylation of the serine-14 residue. GP exists in three interconvertible forms, namely GPa (di-phosphorylated form), GPab (mono-phosphorylated form), and GPb (non-phosphorylated form); however, information on GPab remains scarce. Given the prevailing view that the two GP subunits collaboratively determine their catalytic characteristics, it is essential to conduct GPab characterization to gain a comprehensive understanding of glycogenolysis regulation. Thus, in the present study, we prepared rabbit muscle GPab from GPb, using phosphorylase kinase as the catalyst, and identified it using a nonradioactive phosphate-affinity gel electrophoresis method. Compared with the half-half GPa/GPb mixture, the as-prepared GPab showed a unique AMP-binding affinity. To further investigate the intersubunit communication in GP, its catalytic site was probed using pyridylaminated-maltohexaose (a maltooligosaccharide-based substrate comprising the essential dextrin structure for GP; abbreviated as PA-0) and a series of specifically modified PA-0 derivatives (substrate analogs lacking part of the essential dextrin structure). By comparing the initial reaction rates toward the PA-0 derivative (V derivative) and PA-0 (V PA-0), we demonstrated that the V derivative/V PA-0 ratio for GPab was significantly different from that for the half-half GPa/GPb mixture. This result indicates that the interaction between the two GP subunits significantly influences substrate recognition at the catalytic sites, thereby providing GPab its unique substrate recognition profile.https://doi.org/10.1007/s00726-023-03362-6Chemical probingGlycogen phosphorylaseIntersubunit communicationPhospho–dephospho hybridPhosphorylation regulationSubstrate recognition |
| spellingShingle | Nahori Kamada Ayato Ikeda Yasushi Makino Hiroshi Matsubara Intersubunit communication in glycogen phosphorylase influences substrate recognition at the catalytic sites Amino Acids Chemical probing Glycogen phosphorylase Intersubunit communication Phospho–dephospho hybrid Phosphorylation regulation Substrate recognition |
| title | Intersubunit communication in glycogen phosphorylase influences substrate recognition at the catalytic sites |
| title_full | Intersubunit communication in glycogen phosphorylase influences substrate recognition at the catalytic sites |
| title_fullStr | Intersubunit communication in glycogen phosphorylase influences substrate recognition at the catalytic sites |
| title_full_unstemmed | Intersubunit communication in glycogen phosphorylase influences substrate recognition at the catalytic sites |
| title_short | Intersubunit communication in glycogen phosphorylase influences substrate recognition at the catalytic sites |
| title_sort | intersubunit communication in glycogen phosphorylase influences substrate recognition at the catalytic sites |
| topic | Chemical probing Glycogen phosphorylase Intersubunit communication Phospho–dephospho hybrid Phosphorylation regulation Substrate recognition |
| url | https://doi.org/10.1007/s00726-023-03362-6 |
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