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 (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-spirothiohydantoin (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.

Enzyme Kinetics by Isothermal Titration Calorimetry: Allostery, Inhibition, and Dynamics

“…For example, glycosidase activity can be measured spectrophotometrically with synthetic substrates, such as maltooligomer derivatives with chromogenic chloronitrophenyl (CNP) groups attached (Klaus et al, 1999;Morishita et al, 2000;Ramasubbu et al, 2005). Separate studies on α-amylase and glycogen phosphorylase found the K m values of the fluorogenic substrate analogs to be substantially lower than those of the native substrates determined by ITC, possibly due to interactions of the chromophore with the active site of the enzyme (Lehoczki et al, 2016;Szabó et al, 2019). Thus ITC represents a simple way to accurately characterize how enzymes interact with their biologically relevant molecular partners.…”

Optimization and Validation of an In Vitro Standardized Glycogen Phosphorylase Activity Assay

“…Radioactive measurements have been applied, requiring radioactive labeled substrates and special equipment [ 17 , 27 ]. Other authors have implemented methods to measure the glucose 1-phosphate released from glycogen degradation, using electrospray ionization mass spectrometry [ 28 ], high performance liquid chromatography (HPLC) [ 29 ], or isothermal titration calorimetry (ITC) [ 19 ] methods. However, there are two most frequently applied methods in the literature for measuring GP activity.…”

“…The second method, and most often cited in the literature, evaluates glycogen synthesis, which is based on the colorimetric determination of inorganic phosphate released from glucose 1-phosphate after the glucosyl transfer to glycogen molecule. The first method is not suited for inhibition studies due to potential side interactions between tested compounds and the different enzymes used, phosphoglucomutase and glucose 6-phosphate dehydrogenase [ 19 ]. The second method demonstrates relevant advantages, being less expensive due to the use of only one enzyme, and the formed product is easily detected and allows the straightforward investigation of the inhibitory effect of several compounds.…”

“…Caffeine [ 5 , 17 , 18 , 19 ] and CP-91149 (5-chloro- N -[(1 S ,2 R )-3-(dimethylamino)-2-hydroxy-3-oxo-1-(phenylmethyl)propyl]-1 H -indole-2-carboxamide) [ 16 , 17 ] are well-known allosteric inhibitors of GP. Caffeine was the first natural compound with proved binding to the inhibitor site [ 2 ].…”

“…For instance, besides the above cited isoforms, other enzyme sources including human, pig, rabbit, and rodent, in both the phosphorylated and dephosphorylated state, thereby generate different results [ 7 ]. Since GP catalyzes a reversible reaction ( Figure 1 ) towards glycogen synthesis or breakdown, rather different methods have been additionally described [ 19 ]. Moreover, other experimental conditions also differ, including the concentration of enzyme (when GP is used: 1–5 µg/mL) and substrates (when glucose 1-phosphate is used: 0.5–25 mM and glycogen: 1–20 mg/mL), assay temperatures (22–30 °C), incubation times with the compounds under study (no incubation–15 min), buffers (phosphate, imidazole/HCl, HEPES solution, Tris-malate buffers), and pH (6.8 and 7.2) [ 16 , 17 , 18 , 23 , 24 ].…”