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

• Published in: Carbohydrate research Vol. 477; pp. 58 - 65
• Main Authors: Szabó, Kármen, Kandra, Lili, Gyémánt, Gyöngyi
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ltd 15.05.2019
• Subjects: Activity measurement; Animals; Calorimetry; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - pharmacology; Glycogen phosphorylase; Glycogen Phosphorylase, Muscle Form - antagonists & inhibitors; Glycogen Phosphorylase, Muscle Form - metabolism; Humans; Isothermal titration calorimetry; Kinetics; Muscles - drug effects; Muscles - enzymology; Muscles - metabolism; Rabbits; Reversible enzyme reaction; Structure-Activity Relationship; Substrate inhibition; Reversible enzyme reaction; Substrate inhibition; Activity measurement; Isothermal titration calorimetry; Glycogen phosphorylase
• ISSN: 0008-6215; 1873-426X
• DOI: 10.1016/j.carres.2019.03.014

Glycogen phosphorylase enzymes (GP) catalyse reversible reactions; the glucose transfer from glycogen to inorganic phosphate (Pi, 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/Pi, respectively. Substrate specificity was studied using different maltooligosaccharides and glycogen. Based on the KM 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 Ki 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. Ki 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 Ki 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. [Display omitted] •Reversible enzyme reaction was studied in both directions by ITC.•Substrate specificity of glycogen phosphorylase were determined.•Substrate inhibition was observed on the chromophore containing maltoheptaoside.•Inhibition of forward and reverse reactions resulted in similar inhibition constants.