Question

Q5. The phosphorylase enzyme, which is involved in breakdown of glycogen to glucose, is controlled by both allosteric mechanisms and posttranslational modification. A) Describe how the T to R transition and activity of glycogen phosphorylase would change under the following conditions: i) high AMP levels and activation of phosphoprotein phosphatase, ii) activation of phosphorylase kinase, ili) activation of phosphorylase kinase and high glucose levels. B) What properties would an uncompetitive inhibitor of glycogen phosphorylase have, what would the Lineweaver Burk for this type of inhibitor look like and would increasing the concentration of AMP in the cells overcome this inhibition? C) Draw a Cleland Diagram for the reaction Reaction mechanism is shown below: OH Gel HO HD HO HO- HO CH O Circoger Glycogen O 00 o H O OPO 0- --0 Og o I OH

Answer 1

Ans 1: As given in the question that phosphorylase is regulated by allosteric mean and it exist in two forms Relaxed(R) and Tensed(T) and R form is catalytic active form of phosphorylase kinase because in T form the adjacent amino acids are not phosphorylated; thus the catalysis cannot be carried out.

Phosphorylase exist in two forms T and R. Phosphorylase is allosterically activated by the binding of AMP, AMP activates the phosphorylase by changing its conformation from T to R state. Also AMP is converted to cAMP which activates cAPK and cAPK phsophorylates an inhibitor of phosphoprotein phosphatase which inactivates the phosphatase. So increased AMP level will activate the glycogen phosphorylase and transition of T state into R state takes place.

Phosphorylase and phosphorylase kinase are phosphotransferases. Kinase incorporates phosphoryl group of ATP to the substrate while phosphorylase incorporates the inorganic phosphate into the substrate. So this Phosphorylase is activated by phosphorylation of ser residue in the protein and this is catalyzed by phosphorylase kinase so this means that activation of phosphorylase kinase will increase the activity of phosphorylase enzyme and trasition of T state into R state takes place.

Activation of phosphorylase kinase activates phosphorylase but the binding of glucose to phosphorylase shifts its allosteric equilibrium from the active R form to the inactive T form. So increased level of glucose concentration will decrease the activity of phosphorylase and transition of R state into T state takes place.

2: Uncompetitive inhibitors are those which binds to only enzyme-substrate complex and they decrease both Km and Vmax of the reaction. So the uncompetitve inhibitor of glycogen phosphorylase will only bind to the glycogen and glycogen phosphorylase complex. The Lineweaver Burk plot of this inhibition will have the same slope as uninhibited reaction but this plot's X and Y intercept will be increased.

Uncompetitive inhibitors bind to the enzyme at the same time as the enzyme's substrate. However, the binding of the inhibitor affects the binding of the substrate, and vice-versa.

So binding of AMP will lead to the conformational change in glycogen phosphorylase and its affinity for glycogen will increased as compared to the affinity for inhibitor. Hence the increased concentration of AMP can be used to overcome the inhibition.

3: I am not being able to draw the Cleland diagram, sorry.