Highlights Pentose Phosphate Pathway

Transcription

1 Highlights Pentose Phosphate Pathway 1. The pentose phosphate pathway (PPP) is an interchange of metabolic pathways. 2. It is important to cells as a) an important source of NADPH, b) an important source of ribose-5-phosphate for nucleotide synthesis; c) an interchange; and d) a way to mix and match sugars according to the needs of cells. 3. The cycle only has two oxidations, each of which produces NADPH. It has two isomerizations, and three sets of carbon rearrangements. 4. Carbon rearrangements are catalyzed by transaldolase (one carbon transfers) and transketolase (two carbon transfers). 5. The pathway doesn't really have a beginning or an end and the direction it moves depends on what is available and what the cell needs. 6. Molecules in the highest concentration will serve as input sources for the pathway and molecules in the least abundance will serve as outputs of the pathway. 7. Transketolase requires thiamine pyrophosphate (from the vitamin thiamine) as a coenzyme. 8. Pathways that can be input or output pathways include glycolysis/gluconeogenesis, aromatic amino acid metabolism, and nucleotide metabolism. Highlights Glycogen Metabolism 1. The structure of glycogen consists of units of glucose linked in the alpha 1-4 configuration with branches linked in the 1-6 configuration. 2. Glycogen differs from starch in the amount of branching (much more). 3. Glycogen is a storage form of energy that can yield ATP very quickly, because glucose-1-phosphate can be released very quickly. 4. You should know the function/activites of the enzymes in glycogen breakdown - glycogen phosphorylase, phosphoglucomutase, and debranching enzyme.

2 5. Glycogen phosphorylase action on glycogen yields glucose-1-phosphate. Glycogen phosphorylase exists in two forms - phosphorylase a and phosphorylase b. Phosphorylase a differs from phosphorylase b only in that phosphorylase a contains two phosphates and phosphorylase b contains none. Phosphate is added to glycogen phosphorylase by the enzyme phosphorylase kinase. 6. Glucose-6-phosphate (G6P) has many different fates and sources. First, breakdown of glycogen produces G1P, which is readily converted to G6P. G6P can then go three different directions. In muscle and brain (and most other tissues), G6P enters glycolysis. In liver only, G6P enters gluconeogenesis and is converted to glucose for export to the bloodstream. In other tissues, G6P enters the pentose phosphate pathway and is oxidized to produce NADPH. 7. Breakdown of glycogen by glycogen phosphorylase involves phosphorolysis (use of a phosphate to cleave molecules) instead of hydrolysis. The advantage of this is that the energy of the alpha1-4 bond is used to add phosphate to glucose (forming G1P) instead of using a triphosphate to do so. This saves energy for cells. 8. Glycogen phosphorylase catalyses phosphorolysis of glycogen to within 4 residues of a branch point and then stops. Further metabolism of glycogen requires action of Debranching Enzyme. Debranching enzyme removes three of the remaining four glucoses at a branch point and transfers them to another chain in a 1-4 configuration. The remaining glucose in the 1-6 configuration at the branch point is cleaved in a hydrolysis reaction to yield free glucose. It is the only free glucose released in glycogen metabolism. 9. Phosphoglucomutase interconverts G1P and G6P via a G1,6BP intermediate. The reaction is readily reversible (Delta G zero prime near zero) and the direction of the reaction depends on the concentration of substrates. 10. Synthesis of glycogen is not the simple reversal of the steps in glycogen breakdown. There is an energy barrier that must be overcome - synthesis of the alpha1,4 bond between adjacent glucoses in glycogen. This is accomplished by a 'side-step' reaction that creates uridine diphosphate glucose (UDP-Glucose or UDPG) from G1P. 11. Synthesis of UDPG requires UTP and G1P and produces UDPG and pyrophosphate (PPi). 12. UDPG is an activated intermediate, a molecule with a high energy bond that uses the energy of that bond to donate a part of itself to something else.

3 13. Glycogen synthase catalyzes the addition of a glucose (in an alpha 1-4 linkage) from UDPG. 14. Branching enzyme turns a part of a glycogen chain into a branch by catalyzing breaking of an alpha1-4 bond and moving the piece to another portion of the glycogen and linking it via an alpha 1-6 bond. 15. Regulation of glycogen phosphorylase is by two mechanisms - covalent modification and allosterism. 16. Glycogen phosphorylase is present in two forms that differ in their phosphorylation. GPa (glycogen phosphorylase a) has phosphate and GPb (glycogen phosphorylase b) does not. GPb is converted into GPa by phosphorylation at two sites. Covalent modifications are DIFFERENT from allosteric controls, which interconvert the R and T states of BOTH GPa and GPb. 17. GPb is inhibited by ATP and G6P (converts R state to T state). When the body is at rest, ATP and G6P levels are high enough to turn GPb off. GPb is ONLY converted to the R state (activated) when AMP is present in sufficiently high concentrations. 18. Glucose converts GPa from the R to the T state (inhibition). Nothing is required to convert GPa from the T to the R state, so if glucose is absent (which it usually is), then GPa will be in the R state. 19. Consequently, in normally resting cells, GPa is usually in the R state (active) and GPb is usually in the T state (inactive). Thus, processes like phosphorylation/dephosphorylation which interconvert GPa and GPb have major effects on whether or not glycogen breakdown is occuring. 20. Activation of the epinephrine/glucagon system results in phosphorylation of glycogen synthase (converts glycogen synthase a to b - note there was an inconsistency in the Powerpoint regarding glycogen synthase a and b. I have fixed it, so if you download the current Powerpoint/PDF for this lecture, you'll hve the right nomenclature). 21. The enzyme responsible for phosphorylating glycogen phosphorylase (thus converting GPb to GPa) is known as (glycogen) phosphorylase kinase. This interesting enzyme is activated by two different mechanisms - phosphorylation and/or calcium ions. 22. Calcium ions bind to phosphorylase kinase by virtue of the fact that the protein calmodulin (which binds calcium ions) is a subunit of phosphorylase kinase.

4 23. Note that calcium ions are a factor in muscular contraction. Release of calcium occurs with muscular contraction. Thus, when ATP is needed, phosphorylase kinase is activated by calcium. Activation of phophorylase kinase causes phosphorylation of GPb, forming GPa, favoring glycogen breakdown and, ultimately, ATP production. 24. Calcium alone only partly activates phosphorylase kinase. Full activation of phosphorylase kinase requires that the protein be phosphorylated as well. Phosphorylation of phosphorylase kinase is catalyzed by protein kinase A. Phosphorylation of phosphorylase kinase alone can also partly activate phosphorylase kinase, just as was the case with calcium. Either binding of calcium ion or phosphorylation can happen first. There is no required order to the binding. 25. Phosphorylation of glycogen synthase converts it from glycogen synthase a (dephophosphorylated and active) to glycogen synthase b (phosphorylated and active). Thus, phosphorylation turns OFF glycogen synthase and helps activate glycogen phosphorylase. This is how glycogen metabolism is reciprocally regulated. 26. In the cascade system initiated by binding of epinephrine or glucagon to a cell-surface receptor, turning off the system involves a) inactivating the 7TM (discussed earlier in the term); b) inactivation of the G-protein (GTPase), c) breakdown of camp (phosphodiesterase); and d) reversal of the protein phosphorylations. The last step requires phosphoprotein phosphatase and that enzyme is activated by binding of insulin to the cell surface receptor. 27. Insulin and epinephrine/glucagon work counter to each other. 28. Ephiephrine/glucagon favors phosphorylation of glycogen enzymes, which activates glycogen breakdown and inactivates glycogen synthesis - makes sense when glucose is lacking. 29. Insulin favors dephosphorylation, which activates glycogen synthesis and inactivates glycogen breakdown. 30. In the experiment I showed in class, addition of glucose to purified GPa and GSb caused conversion of GPa to GPb and GSb to GSa. The cause is rooted in the fact that phosphoprotein phosphatase is linked to a protein (called G) that is bound to the GPa when it is in the R state. In this complex of phosphoprotein phosphatase-g-gpa, the phosphoprotein phosphatase is inactive. Glucose can bind allosterically to the R-state of GPa and convert it to the T-state. This change in structure of GPa by binding glucose causes it to release the phosphoprotein phosphatase-g complex. Release of

5 the phosphoprotein phosphatase-g complex causes it to become activated and it dephosphorylates GPa to GPb and GSb to GSa.