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

Kármen Szabó, L. Kandra, G. Gyémánt

Research output: Contribution to journalArticle

Abstract

Glycogen phosphorylase enzymes (GP) catalyse reversible reactions; the glucose transfer from glycogen to inorganic phosphate (P i , phosphorolysis) or the reverse glucose transfer from glucose-1-phosphate (G-1-P) to glycogen (synthesis). Rabbit muscle GPb (rmGPb) was used as a model enzyme to study the reversible enzyme reaction. To follow both directions of this reversible reaction, we have developed a novel isothermal titration calorimetry (ITC) method for the determination of the direct reaction rate. The preference of forward or reverse reaction was ensured by the 0.1 or 10 concentration ratios of G-1-P/P i , respectively. Substrate specificity was studied using different maltooligosaccharides and glycogen. Based on the K M values, glycogen and 2-chloro-4-nitrophenyl maltoheptaoside (CNP-G7) were found to be analogous substrates, which allowed to optimize the method by taking advantage of the CNP chromophore being detectable in HPLC. In case of CNP-G7, substrate inhibition was observed and characterised by K i of 23 ± 7 mM. Inhibition of human GP is a promising strategy for the treatment of diabetes. Our ITC measurements have confirmed that caffeine and glucopyranosylidene-spiro-thiohydantoin (GTH), as known GPb inhibitors, inhibit the rmGPb-catalysed reversible reaction in both directions. K i values obtained in the direction of synthesis (1.92 ± 0.14 mM for caffeine and 11.5 ± 2.0 μM for GTH) have been shown to be in good agreement with the K i values obtained in the direction of phosphorolysis (4.05 ± 0.26 mM for caffeine and 13.8 ± 1.6 μM for GTH). The higher difference between the inhibition constants of caffeine was explained by the non-competitive mechanism. The described ITC method using the developed experimental design and reaction conditions is suitable for activity measurements of different phosphorylase enzymes on various substrates and is applicable for inhibition studies as well.

Original languageEnglish
Pages (from-to)58-65
Number of pages8
JournalCarbohydrate Research
Volume477
DOIs
Publication statusPublished - May 15 2019

Fingerprint

Phosphorylase b
Glycogen Phosphorylase
Calorimetry
Titration
Thiohydantoins
Muscle
Caffeine
Glycogen
Rabbits
Muscles
Enzymes
Substrates
Glucose
Phosphorylases
Chromophores
Medical problems
Substrate Specificity
Design of experiments
Reaction rates
Research Design

Keywords

  • Activity measurement
  • Glycogen phosphorylase
  • Isothermal titration calorimetry
  • Reversible enzyme reaction
  • Substrate inhibition

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry
  • Organic Chemistry

Cite this

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title = "Studies on the reversible enzyme reaction of rabbit muscle glycogen phosphorylase b using isothermal titration calorimetry",
abstract = "Glycogen phosphorylase enzymes (GP) catalyse reversible reactions; the glucose transfer from glycogen to inorganic phosphate (P i , phosphorolysis) or the reverse glucose transfer from glucose-1-phosphate (G-1-P) to glycogen (synthesis). Rabbit muscle GPb (rmGPb) was used as a model enzyme to study the reversible enzyme reaction. To follow both directions of this reversible reaction, we have developed a novel isothermal titration calorimetry (ITC) method for the determination of the direct reaction rate. The preference of forward or reverse reaction was ensured by the 0.1 or 10 concentration ratios of G-1-P/P i , respectively. Substrate specificity was studied using different maltooligosaccharides and glycogen. Based on the K M values, glycogen and 2-chloro-4-nitrophenyl maltoheptaoside (CNP-G7) were found to be analogous substrates, which allowed to optimize the method by taking advantage of the CNP chromophore being detectable in HPLC. In case of CNP-G7, substrate inhibition was observed and characterised by K i of 23 ± 7 mM. Inhibition of human GP is a promising strategy for the treatment of diabetes. Our ITC measurements have confirmed that caffeine and glucopyranosylidene-spiro-thiohydantoin (GTH), as known GPb inhibitors, inhibit the rmGPb-catalysed reversible reaction in both directions. K i values obtained in the direction of synthesis (1.92 ± 0.14 mM for caffeine and 11.5 ± 2.0 μM for GTH) have been shown to be in good agreement with the K i values obtained in the direction of phosphorolysis (4.05 ± 0.26 mM for caffeine and 13.8 ± 1.6 μM for GTH). The higher difference between the inhibition constants of caffeine was explained by the non-competitive mechanism. The described ITC method using the developed experimental design and reaction conditions is suitable for activity measurements of different phosphorylase enzymes on various substrates and is applicable for inhibition studies as well.",
keywords = "Activity measurement, Glycogen phosphorylase, Isothermal titration calorimetry, Reversible enzyme reaction, Substrate inhibition",
author = "K{\'a}rmen Szab{\'o} and L. Kandra and G. Gy{\'e}m{\'a}nt",
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T1 - Studies on the reversible enzyme reaction of rabbit muscle glycogen phosphorylase b using isothermal titration calorimetry

AU - Szabó, Kármen

AU - Kandra, L.

AU - Gyémánt, G.

PY - 2019/5/15

Y1 - 2019/5/15

N2 - Glycogen phosphorylase enzymes (GP) catalyse reversible reactions; the glucose transfer from glycogen to inorganic phosphate (P i , phosphorolysis) or the reverse glucose transfer from glucose-1-phosphate (G-1-P) to glycogen (synthesis). Rabbit muscle GPb (rmGPb) was used as a model enzyme to study the reversible enzyme reaction. To follow both directions of this reversible reaction, we have developed a novel isothermal titration calorimetry (ITC) method for the determination of the direct reaction rate. The preference of forward or reverse reaction was ensured by the 0.1 or 10 concentration ratios of G-1-P/P i , respectively. Substrate specificity was studied using different maltooligosaccharides and glycogen. Based on the K M values, glycogen and 2-chloro-4-nitrophenyl maltoheptaoside (CNP-G7) were found to be analogous substrates, which allowed to optimize the method by taking advantage of the CNP chromophore being detectable in HPLC. In case of CNP-G7, substrate inhibition was observed and characterised by K i of 23 ± 7 mM. Inhibition of human GP is a promising strategy for the treatment of diabetes. Our ITC measurements have confirmed that caffeine and glucopyranosylidene-spiro-thiohydantoin (GTH), as known GPb inhibitors, inhibit the rmGPb-catalysed reversible reaction in both directions. K i values obtained in the direction of synthesis (1.92 ± 0.14 mM for caffeine and 11.5 ± 2.0 μM for GTH) have been shown to be in good agreement with the K i values obtained in the direction of phosphorolysis (4.05 ± 0.26 mM for caffeine and 13.8 ± 1.6 μM for GTH). The higher difference between the inhibition constants of caffeine was explained by the non-competitive mechanism. The described ITC method using the developed experimental design and reaction conditions is suitable for activity measurements of different phosphorylase enzymes on various substrates and is applicable for inhibition studies as well.

AB - Glycogen phosphorylase enzymes (GP) catalyse reversible reactions; the glucose transfer from glycogen to inorganic phosphate (P i , phosphorolysis) or the reverse glucose transfer from glucose-1-phosphate (G-1-P) to glycogen (synthesis). Rabbit muscle GPb (rmGPb) was used as a model enzyme to study the reversible enzyme reaction. To follow both directions of this reversible reaction, we have developed a novel isothermal titration calorimetry (ITC) method for the determination of the direct reaction rate. The preference of forward or reverse reaction was ensured by the 0.1 or 10 concentration ratios of G-1-P/P i , respectively. Substrate specificity was studied using different maltooligosaccharides and glycogen. Based on the K M values, glycogen and 2-chloro-4-nitrophenyl maltoheptaoside (CNP-G7) were found to be analogous substrates, which allowed to optimize the method by taking advantage of the CNP chromophore being detectable in HPLC. In case of CNP-G7, substrate inhibition was observed and characterised by K i of 23 ± 7 mM. Inhibition of human GP is a promising strategy for the treatment of diabetes. Our ITC measurements have confirmed that caffeine and glucopyranosylidene-spiro-thiohydantoin (GTH), as known GPb inhibitors, inhibit the rmGPb-catalysed reversible reaction in both directions. K i values obtained in the direction of synthesis (1.92 ± 0.14 mM for caffeine and 11.5 ± 2.0 μM for GTH) have been shown to be in good agreement with the K i values obtained in the direction of phosphorolysis (4.05 ± 0.26 mM for caffeine and 13.8 ± 1.6 μM for GTH). The higher difference between the inhibition constants of caffeine was explained by the non-competitive mechanism. The described ITC method using the developed experimental design and reaction conditions is suitable for activity measurements of different phosphorylase enzymes on various substrates and is applicable for inhibition studies as well.

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KW - Isothermal titration calorimetry

KW - Reversible enzyme reaction

KW - Substrate inhibition

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