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

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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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). https://doi.org/10.1007/s10719-017-9776-5

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Keywords

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