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 (SG, SG, SG, SG, SG, 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 SG 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 -AltNAc-Glc -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 SG -SG subsites was important for the catalysis of GP. By comparing the initial reaction rate toward the PA-0 derivatives (V ) with that toward PA-0 (V), we found that the value of V /V 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.

Fragmentation of Oligosaccharides from Sodium Adduct Molecules Depends on the Position of <i>N</i>-Acetyl Hexosamine Residue in Their Sequences in Mass Spectrometry

“…ese spectra also di ered from the spectra produced from the protonated molecules [M+H] + , in which all the product ions were generated at the reducing-end side, with PA predominating in all compounds 1 to 6 because the PA amine group was strongly protonated as the charge center. 13) In the product ion spectra of the sodium adducts [M+Na] + (Fig. 2), the ion peaks labeled by a red circle contained a PA reducing end with the HexNAc residue, and they were generated by the sequential removal of the glucose (Glc) residues from the non-reducing terminal such as 5. e peaks labeled by a red inverted triangle had a PA reducing end without the HexNAc residue, and they were generated by the removal of the Glc and HexNAc residues from the non-reducing terminal (Figs.…”

“…1), was prepared chemically from 3A-amino-3A-deoxy-(2AS,3AS)-α-cyclodextrin (Tokyo Chemical Industry Co., Ltd., Tokyo), as described in our previous paper. 13)…”

“…In this study, we systematically prepare six maltohexasaccharides containing one HexNAc residue, in which the sequential position of the HexNAc residue di ered. 13) We reveal the in uence of the HexNAc position on the sequential analysis of oligosaccharides.…”

Studies on the reversible enzyme reaction of rabbit muscle glycogen phosphorylase b using isothermal titration calorimetry

“…Ki values of 11.3 μM and 13,8 μM were calculated from Ki* using Equation (5), where [S]=5 mg/ml and KM=1.5 mg/ml were applied for glycogen (Table 2).…”

“…There is an additional option for the measurement of G-1-P released from glycogen, namely the use of an anion-exchange HPLC method on Carbopack PA10 column with pulsed amperometric detection [4]. Fluorescent substrates (pyridylaminated maltohexaose and some of its derivatives) and HPLC measurement on polymer based HILIC column have recently been utilized for probing the catalytic site of rabbit muscle GPb (rmGPb) [5].…”