This experiment investigated the kinetics of the enzyme glycogen phosphorylase b which is important to metabolism. AMP is an allosteric activator of the enzyme because it converts glycogen phosphorylase b from its T state to the R state which is the active form. Caffeine is an inhibitor because it binds the nucleoside inhibitor site. When it binds this site, it stabilizes the inactive T state and blocks the catalytic site which needs to be open for enzyme activity to occur.
The glycogen phosphorylase b was purified with hydrophobic column chromatography and the concentration was determined with a Bradford Assay. The kinetics of glycogen phosphorylase b were studied by finding a molar extinction coefficient, 0. 1617 mM cm-1 A-1, for inorganic phosphate that could be used to determine the concentration of inorganic phosphate produced by enzyme activity. The optimal condition for the enzyme was determined through thermal inactivation and a range assay for the concentration of enzyme. The optimal condition was 30 to 40 degrees at a sixteen-fold dilution.
The optimal condition was used in addition to the standard curve for inorganic phosphate to determine the activity of glycogen phosphorylase b in the presence of the llosteric activator, AMP, and inhibitor, caffeine. Introduction Understanding how glycogen phosphorylase functions is important to understanding metabolism in cells. Glycogen phosphorylase plays a role in the degradation of glycogen to glucose. It produces gluose-1-phosphate in the muscle that will be converted to glucose-6-phosphate to be used by glycolysis to produce the needed energy for contraction for muscle (Johnson).
The actions of the phosphorylase are important to the regulation of glucose levels in the cells. The activity of the glycogen phosphorylase is effected by regulators. These regulators effect the activity of the phosphorylase but not the actual reaction. Regulators bind allosteric sites in order to effect the enzyme activity usually through conformation changes. The activity can either be increased or decreased depending on whether the regulator is an effector or inhibitor for the enzyme. An important allosteric effector for glycogen phosphorylase is AMP.
AMP will bind an allosteric site with high affinity to convert the glycogen phosphorylase from a T (inactive) form to the R (active) form of the enzyme. Once the enzyme is activated by AMP it can assist n the degradation of glycogen. Glycogen phosphorylase can also be inhibited by regulators. One inhibitor of the enzyme is caffeine. Caffeine is a related heterocyclic ring compound that will bind the nucleoside inhibitor site of the enzyme. It has a dual action of stabilizing the inactive T form of the enzyme and blocking the catalytic site needed for activation.
Caffeine works in concert with glucose so it will promote the binding of glucose and vice versa. Caffeine and glucose act as inhibitors for both glycogen phosphorylase a and b (Johnson). In order to properly study glycogen phosphorylase b, it must irst be purified. Hydrophobic column chromatography was used in order to achieve this. The glycogen phosphorylase b protein binds the solid hydrophobic resin that is added to the column. The undesired proteins will be washed away in flow through and earlier fractions when decreasing concentrations of ammonium sulfate.
The fractions can then be analyzed by gel electrophoresis to see if the desired protein was isolated. Glycogen phosphorylase is a homodimer with 97-kDa subunits. A band should appear around 97 kDa indicating the presence of glycogen phosphorylase b. The bands are compared to a protein ladder (Life Technologies). Once the protein is isolated and purified, enzymatic assays are run to determine the kinetic constants. Kinetic constants are a good way to compare the effects of regulators on the enzyme. These kinetic constants include Vmax and Km. Vmax represents the maximal reaction rate and is the plateau of a Michaelis- Menten curve.
At this point, the active sites are saturated or no longer as accessible due to conformational change. The Km is the Michaelis constant and is an inverse of the substrate’s affinity for the binding site. As an inverse, a lower Km represents a higher substrate affinity for the enzymatic binding site. Inhibitors should result in a lower Vmax due to their effects on the activity of the enzyme, while effectors should increase the enzymatic activity. The kinetic assays studied the activity of glycogen phosphorylase b when the AMP activator is added and when an inhibitor, like caffeine, is added.
The protein was isolated and purified and the concentration was found using a Bradford Assay. For the assays, an inorganic phosphate standard curve was created to discover the molar extinction coefficient for norganic phosphate that is produced from the enzyme activity. This coefficient was used to determine the concentration of inorganic phosphate produced in assays analyzing the effect of temperature inactivation and enzyme concentration on the activity. Once the optimal conditions were found, the effect of activator AMP concentration and the presence of caffeine inhibitor was analyzed through similar assays.
Results and Discussion After the protein was isolated using hydrophobic column chromatography, the fractions were analyzed with gel electrophoresis. A protein ladder was used in lane one for eight comparisons for the remaining lanes (Figure 1). The reaction buffer had been leaking from the gel initially, so a slight curvature can be seen and the protein ladder lane bled into the second lane which contained the protein sample. Three protein bands were visualized on the protein sample. The band containing the glycogen phosphorylase b was the band right below the 98-kDa orange band.
This was indicative of the glycogen phosphorylase b because the enzyme has a mass of 97-kDa. Most of the glycogen phosphorylase b from the column chromatography can be seen in fifth fraction which was in the th lane on the gel. There was also a little protein present in the fourth and sixth fractions. This indicated that the glycogen phosphorylase eluted off with a lower ammonium sulfate concentration. The protein concentration of the chromatography fractions was then determined. The absorbance of each fraction was read at 595 nm.
Bovine gamma globulin is the standard protein for Bradford Assays. The standard curve for bovine gamma globulin shows that each absorbance unit is equivalent to 1. 5 mg/mL. This value was applied to the absorbance values for the chromatography fractions to calculate the concentration of lycogen phosphorylase b in the fractions. Table 1 shows the calculated concentrations. Fraction 5 had the highest amount of protein in all the fractions collected from the column. After the protein was purified, studies on the activity of the enzyme were conducted.
Inorganic phosphate is produced from glycogen phosphorylase activity, so the presence of inorganic phosphate is indicative of enzyme activity. First, a standard curve for inorganic phosphate was created in order to find the molar extinction coefficient. The absorbance was measured for varying amounts of inorganic phosphate (Table 3). A graph was created for the concentration of inorganic phosphate versus the absorbance value (Figure 2). Linear regression was then used to analyze the data and find a slope showing the concentration of inorganic phosphate per unit of absorption. This slope was found to be 0. 1617 (Table 4).
The slope represented the molar extinction coefficient because the units were mM/unit of absorbance and Beer’s Law indicates that molar extinction coefficients are concentration per unit of absorbance per path length. The path length was 1 cm, so the slope divided by 1 cm rovided the molar extinction coefficient. This value represented the molar extinction coefficient for inorganic phosphate and could be used on new assays to calculate the concentration of inorganic phosphate from glycogen phosphorylase b. Optimal activity of glycogen phosphorylase was then studied through assays for thermal inactivation and a range of enzyme concentrations.
For thermal inactivation, the enzyme was treated to varying degrees of temperature before being used with the assays. The absorbance values for the different concentrations were recorded in Table 6. The absorbance values ere adjusted based on the value of the blank. The amount of absorbance was then visualized on a bar graph shown in Figure 4. The level of absorbance started to drop at 50 degrees and severely dropped at 60 and 70 degrees indicating that the enzyme was inactivated at temperatures of 50 degrees and higher.
At high temperatures, the intermolecular bonds for protein folding may denature and decrease enzyme activity. There was also a slightly lower absorbance for the untreated enzyme. At too low of temperatures, the activation barrier may be too difficult to overcome without enough energy. The ptimal temperature was determined to be between 30 and 40 degrees. The next optimization step was the range of enzyme concentration that was the most effective. In this experiment, a sixteen-fold dilution of enzyme was studied. This dilution was tested for five different reaction times ranging from two to ten minutes.
The absorbance values were recorded in Table 7 and graphed in Figure 5. Figure 5 showed that there was a linear increase in activity from a two minute reaction time to a six minute reaction time. After six minutes there was an increase in the slope. This graph was compared to the graphs of other nzyme concentrations and was determined to be the most linear and optimal concentration for glycogen phosphorylase b. A linear relationship is the most indicative of an optimal dilution. The data for the other dilutions were not shared in the lab so they were not reported here.
Once the optimal conditions were discovered, the kinetics of the enzyme were studied. The Vmax and Km were discovered through experiments with the allosteric activator AMP. First an assay without any enzyme was completed to determine what amount of inorganic phosphate was present not from enzyme activity. Table 8 shows the concentration of inorganic phosphate calculated for each concentration of glucose-1-phosphate substrate. The linear regression was then calculated and had a slope of 0. 03 mM inorganic phosphate per mM glucose-1phosphate.
Table 9 shows the amount of inorganic phosphate present in the assay that was not created from enzyme activity Assays on varying concentrations of AMP were then completed. Figure 6 shows that the assay without any AMP had the lowest Vmax and Km indicating that the enzyme was not as active without any AMP present. Table 15 compares the Vmax and Km found for the varying concentrations of AMP. The concentration of inorganic phosphate was corrected with data found in Table 9 by subtracting the amount of inorganic phosphate not from enzyme activity. As expected, the Vmax for 0. 5 mM AMP was the next lowest at 1. 19 mM/min.
It was expected that the Vmax would increase as the concentration of AMP increased, but 4 mM AMP had a smaller Vmax than 1 mM or 2 mM AMP at 1. 361 mM/min. The 1 mM AMP concentration had the highest Vmax at 1. 455 mM/min. This would suggest that a high concentration of AMP could start to inhibit the enzyme rather than activate it or there was an error in the assay. All the Michaelis-Menten curves did not have the standard curve seen suggesting the kinetics were not truly following MichaelisMenten. This would lead to a misinterpretation of how the concentration of AMP effected the enzyme.
Finally, assays were run to study the effect of caffeine on the glycogen phosphorylase b. Caffeine is an inhibitor so a decrease in inorganic phosphate should be seen as the concentration of inhibitor increases. Figure 7 shows that there was a decrease in absorbance, and thus a decrease in activity when there was inhibitor present, however there was no difference in activity between the varying amounts of caffeine. A possible reason for this could be that the concentration of inhibitor was too low to have a greater effect on the activity of the glycogen phosphorylase
b. Procedure Glycogen Phosphorylase b Purification Glycogen Phosphorylase b was purified using hydrophobicity chromatography. A column was prepared with a well-suspended 70% slurry of butyl-sepharose as the resin. The contaminating protein was removed with five aliquots of HEG pH 7. 0 buffer. HEG buffer contained 20 mM HEPES PH 7. 0, 0. 1 mM Disodium EDTA, 10% (v/v) glycerol, and 1 mM dithiothreitol. The column was prepared to bind the protein by equilibrating the column with five aliquots of 100% BAS. 100% BAS was prepared from 1. 1 Mammonium sulfate in HEG.
500 microliters of 0. 5 mg/ml glycogen phosphorylase b was added to the column. The eluent from this step was collected in a “flow-thru” tube. The resin was then washed with two aliquots of 100% BAS. The eluent from the first aliquot was collected in the “flow-thru” tube. The eluent from the second aliquot was collected in a “wash” tube. BAS with decreasing amounts of ammonium sulfate from 80% to 0% by 20% increments. Each eluent was collected in a separate tube. A final aliquot of HEG was added and the eluent was collected in a new tube.
The samples from the tubes with the eluents were applied to gel electrophoresis along with a SeeBlue Plus2 protein ladder. A sample buffer of 45 mL DI water, 30 mL 0. 5 M DTT, and 75 mL 4X LDS buffer was created. An equal portion of sample buffer was added to each of the collected samples from the column collections in addition to the glycogen phosphorylase b sample. The mixtures were spun and heated at 70 degrees for ten minutes. The samples were then introduced to the gel and ran at 180 volts. Once the gel finished running, it was stained in Instant Blue protein stain on a rocker and later visualized on a light box.
A Bio-Rad Protein Assay was ran for the samples collected from the column and the glycogen phosphorylase b sample. After 50 microliters of sample were mixed with 2. 5 mL of 1X Dye Reagent, they were left standing for 5 minutes and then ran at 595 nm. The blank contained only 1x Dye Reagent in order to zero the instrument. The concentration of protein was calculated using the standard bovine gamma globulin which is 1. 5 mg/mL per unit of absorption. Column Fractions, Thermal Inactivation, and Enzyme Titration