Abstract
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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Abbreviations
- AltNAc:
-
3-acetoamido-3-deoxy-D-altrose
- CD:
-
Cyclodextrin
- CP-91149:
-
[R-(R*,S*)]-5-chloro-N-[3-(dimethylamino)-2-hydroxy-3-oxo-1-(phenylmethyl)propyl]-1H-indole-2-carboxamide
- DHB:
-
2,5-dihydroxybenzoic acid
- GDE:
-
Glycogen debranching enzyme
- Glc:
-
D-glucose
- Glc-1-P:
-
α-D-glucose 1-phosphate
- GlcPA:
-
1-deoxy-1-[(2-pyridyl)amino]-D-glucitol
- GP:
-
Glycogen phosphorylase
- HPLC:
-
High-performance liquid chromatography
- MALDI-TOF MS:
-
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
- MW:
-
Molecular weight
- α-NH2-CD:
-
3A-amino-3A-deoxy-(2AS,3AS)-α-cyclodextrin
- PA:
-
Pyridylamino
- Pi :
-
Inorganic phosphate
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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). https://doi.org/10.1007/s10719-017-9776-5
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DOI: https://doi.org/10.1007/s10719-017-9776-5