The R-subunit would not the able to release the catalytic subunit, so this mutant of protein kinase A would be incapable of being activated.

Transcription

1 1. Explain how one molecule of cyclic AMP can result in activation of thousands of molecules of glycogen phosphorylase. Technically it takes four molecules of cyclic AMP to fully activate one molecule of protein kinase A, although partial activation is possible with one molecule. This causes release of active Protein kinase A (catalytic) subunit. Protein kinase A then activates on phosphorylase b kinase, but not just on a one to one molecular basis. Protein kinase A is an enzyme that acts by transferring phosphate, from ATP as donor, to Ser or Thr side chains in the target sequence of the substrate, phosphorylase b kinase. This is a catalytic reaction, so one molecule of protein kinase A may be able to act on molecules of phosporylase b kinase. When phosphates are added to the α and β subunits of phosphorylase b kinase, this unmasks the catalytic site in the γ subunit. Phosphorylase b kinase recognizes the phosporylase b form of glycogen phosphorylase as a substrate, and transfers phosphate from ATP to Ser 14 of the phosphorylase b, converting it into phosphorylase a. This is also a catalytic reaction, so each molecule of phosphorylase b kinase can act on molecules of phosphorylase b. The combined effect of the two catalytic steps is capable of inducing activity in molecules of glycogen phosphorylase (phosphorylase a) per molecule of protein kinase A activated. When a signal is communicated by a series of catalytic steps, this is called a cascade. A cascade of catalytic steps can amplify a weak input signal. Hormone signals can be weak because the hormone is dispersed throughout the body. 2. What effect might you expect to result from the following mutations in protein kinase A? a) a mutation that prevents cyclic AMP from binding to the R subunit The R-subunit would not the able to release the catalytic subunit, so this mutant of protein kinase A would be incapable of being activated. b) a mutation that prevents R subunit from binding to C-subunit If the R subunit can't bind to the C subunit, the kinase would be permanently active, and would not change activity in response to camp. c) a mutation that increases the binding affinity of R and C subunits If R subunit bound more tightly to C subunit, it might take a higher concentration of camp to dissociate the subunits. For a given concentration of camp, the degree of activation observed would be less. d) a mutation that prevents ATP from binding to C subunit. ATP is the phosphate donor. There would be no catalytic activity for this mutant.

2 3. What is the rationale for positive regulation of glycogen phosphorylase and mammalian phosphofructokinase 1 by AMP? Most reactions that consume ATP do so by removing a single phosphate, yielding ADP as a product. ADP is the substrate the ATP synthase in oxidative phosphorylation, so at first glance, AMP seems to be out of the picture. However, the concentration of AMP is related to the [ATP] and [ADP] levels through the action of myokinase, which is normally at equilibrium: ATP + AMP Ö ADP + ADP K eq = 2.2 [ADP] 2 K eq = [ATP][AMP] rearranges to [AMP] = [ADP] 2 K eq [ATP] Since [AMP] is proportional to the square of [ADP], this means that a doubling of [ADP] should result in at least fourfold increase in [AMP] (more since [ATP] decreases as [ADP] increases). [AMP] is extra sensitive to changes in cellular energy status. 4. Which amino acids are the targets for protein kinase action? Ser and Thr are the most common (Protein kinase A, phosphorylase b kinase, cyclin dependent kinases), followed by Tyr. The phosphate attaches to Ser, Thr or Tyr as a phosphate ester. Phosphohistidine and phosphoarginine (phosphoamides) are less common in mammalian systems, but His does occur in bacterial cells. Both are high energy phosphates. 5. What might be the effect of the following mutations in glycogen phosphorylase? a) ser 14 changed to alanine When alanine replaces Ser in a phosphorylation target sequence, the alanine side chain can't be phosphorylated. Since the effect of phosphate is largely due to its negative charge, and alanine is a neutral amino acid, the mutation can't be activated by protein kinase. The site of Ser 14 is distant from the catalytic site, so the mutant would function as for phosphorylase b, but would not be able to be converted to phosphorylase a. Effectors such as AMP or glucose-6-phosphate would still have the same ability to direct the allosteric change from T to R state or vice versa. b) ser 14 changed to aspartate When aspartate replaces Ser in a phosphorylation target sequence, the aspartate side chain can't be phosphorylated can't be phosphorylated by a serine-directed protein kinase either. Since the effect of phosphate is largely due to its negative charge, and aspartate is negative at normal ph, the mutation will behave as permanently activated. However, phosphate is -PO 4 2, whereas aspartate only carries a single negative, so the activation effect is only partial, and the enzyme would behave as phosphorylase a, but with slightly less tendency towards R state than the Ser-PO 4 2 version of phosphorylase a, and more susceptibility to negative effector.

3 6. What is the difference between positive regulation and positive feedback? Negative regulation and negative feedback? Positive regulation refers to regulation by an effector that increases catalytic activity or substrate affinity. (Affinity is reciprocally related to K M of an enzyme or P 50 for gas-binding protein such as hemoglobin.) This may be by a cooperative allosteric mechaism, or by phosphorylation or by proteolytic cleavage. In the case of cooperative allosteric regulation, positive regulation increases the tendency to be in high-affinity R state. Positive regulators are substances that suggest that the product of the enzyme is needed,.e.g. presence of excess ATP suggests that ATCase should start making more pyrimidines. Negative regulation refers to regulation by an effector or inhibitor that decreases activity or substrate affinity. In the case of cooperative allosteric regulation, negative regulation increases the tendency to be in low-affinity T state. Negative regulators are substances that suggest that the product of the enzyme is not needed,.e.g. presence of excess ATP suggests that PFK1 should not allow glycolysis to continue so as to not waste substrate. Presence of CTP suggests that the cell has enough pyrimidines, and does not need to expend energy and substrates by allowing ATCase to continue making pyrimidines. Negative regulators of this type are frequently end products of the enzyme or pathway. Feedback always involves regulation, but regulation is not necessarily feedback. Feedback refers to the effects that occur when an end product, sometimes of a single reaction but more usually a pathway, acts as the regulator. Positive feedback occurs when an end product X of an enzyme or pathway stimulates the activity of that pathway. More X is made, so that more stimulation occurs, so that even more X is made, in an escalating trend. This will continue until the available starting material or substrate is consumed. This is desirable when one wishes to switch quickly from one behaviour to another, from totally off to maximum on. e.g. small amounts of trypsin activated by enteropeptidase activate more trypsin, this activates even more, and so on until no trypsinogen remains to be converted to active form. This is positive regulation that is also positive feedback. When ATCase speeds up due to high ATP levels, this is positive regulation but not positive feedback, because ATP is not the end product of a pyrimidine synthesis pathway. Negative feedback refers to end product inhibition of a pathway, which is an effective way to maintain a constant levels of the end product somewhere between extreme max and extreme minimum. If some of the product disappears because it is used, the drop in concentration causes a speed-up in the pathway rate to replace it. If the concentration of the end product starts to build up, negative feedback slows down production to bring things back to balance. CTP is a negative regulator of ATCase, and provides negative feedback control, because presence of CTP (cytidine triphosphate) indicates that enough pyrimidine is currently available.

4 Would you describe the following regulatory processes as positive or negative regulation and involving positive or negative feedback? Regulation Feedback a) regulation of ATCase by CTP negative negative b) regulation of phosphofructokinase by ATP negative negative c) regulation of phosphofructokinase positive not feedback by fructose-2,6-bisphosphate Fructose-2,6-bisphosphate is not an end product of the enzyme or pathway d) regulation of cyclin dependent kinase positive positive by its specific phosphatase e) regulation of cyclin dependent kinase +ve for Thr positive by self-phosphorylation f) activation of chymotrypsinogen positive not feedback Chymotrypsin is not responsible for the activation step in conversion of chymotrypsinogen to p-chymotrypsin g) the blood clotting cascade positive positive 7. What kind of chemical bonding occurs as blood clots? What is the difference between a soft clot and a hard clot? Fibrin molecules first assemble due to electrostatic bonds. Subsequently they become cross-linked by forming side chain amide bonds (covalent) between glutamine on one chain and lysine on a neighbour. 8. Distinguish the roles of the following blood components: prothrombin, thrombin, fibrinogen, fibrin, plasminogen, plasmin. Prothrombin is the inactive proenzyme of thrombin, which is a serine protease that targets the sequence Arg-Gly, cutting the bond after Arg. The role of thrombin is to split off the fibrinopeptides A and B from fibrinogen. Prothrombin has an unusually large propeptide that is essential for rapid activation, since clot formation usually occur on a time scale of minutes. This propeptide contains the modified amino acid γ-carboxyglutamate, which enhances Ca 2+ binding by prothrombin. When prothrombin binds Ca 2+, this helps co-localize prothrombin with its activating factor Xa. Fibrinogen is a giant (340 kda) precursor of fibrin. It is made up of three chains, Aα, Bβ and γ which are largely a-helical, and wind around each other in a coiled-coil, so that the molecule is fibrous in shape. Thrombin clips off the fibrinopeptides A and B (18-20 amino acids each) to make fibrin, and this unmasks binding sites at the midpoint of fibrin that bind and recognise the end domains of the fibre. As a result, fibrin molecules assemble in a brick-like pattern to form giant fibres that cause blood to gel and clot. Plasminogen is the proenzyme form of plasmin, which is another serine protease that specifically cuts fibrin fibres in blood clots. This allows clots to dissolve, a process that normally takes place on a time scale of days.

5 9. Why does injection of tissue plasminogen activator within the first 30 minutes following heart attack or stroke frequently prevent long term damage? Tissue plasminogen activator is an enzyme that is normally present in trace amounts in blood. It converts plasminogen into plasmin, so that blood clots can dissolve. Normally this is a slow process. By injecting extra tissue plasmin activator, the intention is to stimulate activation of more clot-dissolving plasmin so that blood vessel blockages that caused the abnormal condition can be cleared. This is most successful if the fibrin clot is still in the early soft-clot stage. After about 30 minutes, Clotting factor XIII starts to produce covalent crosslinks between fibrin molecules by linking up glutamine side chains on one strand with lysine sidechains on another, to form sidechain amide bonds. The resulting hard clot is much more difficult to dissolve.