(8/25/10) - Glycolysis and TCA Cycle

Please see: Introduction to Metabolic Pathways =Glycolysis:=

'1. Outline the sequence of reactions in aerobic and anaerobic glycolysis. Mark on the pathway the regulatory enzymes. '

'2. Summarize the energetics of aerobic and anaerobic glycolysis by highlighting the reactions involved in the utilization and generation of ATP, and the net yield of ATP during glycolysis (include those reactions that generate NADH).  '

Glycolysis: 2 ATP used ( 1 in the D-glucose ==> glucose-6-P reaction and another in the fructose-6-P ==> fructose 1,6-bisP reaction), 2 NADH produced (1per glyceraldehydes-3-P ==> 1,3 biphosphoglycerate reaction), 4 ATP produced (2 per PEP ==> pyruvate reaction) - this yields a net total of 2 ATP and 2 NADH or the equivalent of 8 ATP - if pyruvate ==> lactate, the NADH generated by glycolysis is used to reduce pyruvate to lactate, and the net yield is 2 moles of ATP per molecule of glucose converted to lactate

'3. Notate the net reaction for the conversion of glucose into pyruvate and the number of ATP and NADH molecules formed.  '

Glucose + 2 P + 2 ADP + 2 NAD+ --> 2 pyruvate + 2ATP + 2NADH + 2 H+ + 2H2O

'4. Identify the major regulatory step of glycolysis and describe in detail the mechanism(s) of regulation of the enzyme(s) involved with particular reference to the production or conversion of 2,6-bisphosphate. Compare and contrast the regulatory events in the fed vs. fasting states.  '

The major regulatory step of glycolysis is conversion of fructose-6-P ==> fructose-1,6-bisP. PFK-1 is the enzyme that catalyzes this reaction, and it is regulated in numerous ways. Its synthesis is increased in response to insulin, it is allosterically inhibited by high levels of ATP and citrate, and it is stimulated by AMP and fructose-2,6-bisP. When insulin is high, some of fructose-6-P is diverted to make fructose-2,6-bisP, and this reaction is catalyzed by a protein with two domains (PFK-2 and FBP-2). Fructose-2,6-bisP stimulated PFK-1 by increase PFK-1’s affinity for its subtrate while simultaneously inhibiting FBP (the enzyme that catalyzes the reverse reaction, fructose-1,6-bisP ==> fructose-6-P). When glucagons levels are high and insulin levels low, PKA phosphorylates PFK-2 and in doing so, inhibits production of fructose-2,6-bisP. Under these conditions, PFK-1 is not stimulated and FBP is not inhibited, thereby allowing the reverse reaction to proceed.

5. Identify the step(s) in glycolysis:  

D-glucose ==> glucose-6-P ==> fructose-6-P ==> fructose-1,6-bisP ==> 2 (glyceraldehyde-3-P) ==> 2 (1,3 biphosphoglycerate) ==> 2 (3 phosphoglycerate) ==> 2 (2 phosphoglycerate) ==> 2 (phosphoenolpyruvate) ==> 2 (pyruvate)

'A. That illustrate the use of coupled reactions to drive thermodynamically unfavored processes including substrate level phosphorylation.  '

D-glucose ==> glucose-6-P is coupled with the dephosphorylation of ATP ==> ADP

Fructose-6-P ==> fructose -1,6-bisP is coupled with the dephosphorylation of ATP ==> ADP

2 glyceraldehyde-3-P ==> 1,3 biphosphoglycerate is couple with NAD+ ==> NADH

1,3 biphosphoglycerate ==> 3 phosophoglycerate is couple with ADP ==> ATP

phosphoenolpyruvate ==> pyruvate is coupled with ADP ==> ATP

'B. That exhibit feed-forward regulation.  '

2 phophoenolpyruvate ==> 2 pyruvate catalyzed by pyruvate kinase - pyruvate kinase is stimulated by fructose-1,6-bisP in an example of feed-forward regulation

'C. Whose product can be diverted to produce an important regulator of oxygen binding to hemoglobin. Note the physical characteristics of this molecule and understand how these enable the molecule to perform its function.  '

Glyceraldehydes-3-P ==> 1,3 bisphosphoglycerate In RBCs, 1,3-bisphophoglycerate can be converted to 2,3-biphosphoglycerate (BPG), a compound that decreases the affinity of hemoglobin for oxygen.

'D. That have clinical significance, including the most common genetic deficiencies and their consequences.  '

Pyruvate dehydrogenase deficiency: decreases ability to convert pyruvate into acetyl CoA, causing problems for the brain which is dependent upon TCA for most of its enery needs Pyruvate kinase deficiency: leads to decreased glycolysis, and in erythrocytes leads to hemolytic anemia

'6. List the functional differences between hexokinase and glucokinase and identify when they are operational during the feed-fast cycle. Explain how this occurs i.e. how are they regulated. Understand why phosphorylation is an important first step in the glycolytic pathway.  '

Hexokinase and glucokinase both catalyze the reaction of D-glucose ==> glucose-6-P. Glucokinase works in the liver and in beta cells while hexokinase is found in other cells. Glucokinase synthesis is increased in response to high insulin levels and decreased in response to high glucagons levels, and it is inhibited by fructose-6-P. Hexokinase is inhibited by G-6-P. Additionally, hexokinase has a low Km for glucose, meaning that is often working near its maximum rate even when blood glucose levels are low. Glucokinase, on the other hand, has a high Km for glucose, meaning that it is very active when glucose levels in the hepatic portal vein are high and it is inactive when glucose levels are low.

'7. Explain why glycolysis is essential for normal RBC functions including consequences in deficiencies in glycolytic enzymes and the role of glycolysis in adaption to high altitude.  '

'8. Describe how flux down or up the pathway is coordinated by hormonal regulation.  '

'9. Describe the role of lactate production and the clinical significance of lactic acidosis.  '

Pryruvate is converted to lactate when oxygen is not present and it is couple to the oxidation of NADH to NAD+. By regenerating NAD+, glycolysis can continue. Lactic acidosis results from emergency anerobic glycolysis and refers to elevated lactate levels in the plasma.

'10. Review the uptake of glucose into cells.  '

Glucose cannot enter cells by diffusion. Cells of the intestinal mucosa transport glucose against a gradient in an energy dependent process that is driven by the sodium gradient across the membrane. Transport in some tissues (brain, liver, erythrocytes, adipose, muscle) is by carrier-mediate diffusion and is driven by a concentration gradient. Transporters GLUT1-GLUT5 have two variable conformations: glucose binds, alters conformation, and is then released into the cell. GLUT4 is found in skeletal muscle and adipose tissue, and its presence in the membrane is increased by insulin.

=TCA:=

'1. Outline the sequence of reactions in the TCA cycle and mark on the pathway the enzymes involved and which steps of the cycle yield CO2, NADH, FADH2 and GTP.  '

Pyruvate ==> acetyl CoA

Acetyl CoA ==> citrate ==> isocitrate ==> alpha ketoglutarate ==> succinyl CoA ==> succinate ==> fumurate ==> L-malate ==> oxaloacetate ==> citrate

Pyruvate dehydrogenase catalyzes pyruvate ==> acetyl CoA

Citrate synthase catalyzes acetyl CoA ==> citrate

Isocitate dehydrogenase catalyzes isocitrate ==> alpha ketoglutarate

Alpha ketoglutarate dehydrogenase catalyzes alpha ketoglutarate ==> succinyl CoA

Pryuvate ==> acetyl CoA: NADH + CO2

Isoctirate ==> alpha ketoglutarate: NADH + CO2

Alpha ketoglutarate ==> succinyl CoA: NADH + CO2

Succinyl CoA ==> succinate: GTP

Succinyl CoA ==> fumurate: FADH2

L-malate ==> oxaloacetate: NADH

'2. Identify the metabolic sources of the two intermediates required in the first step of the TCA cycle.  '

Actetyl CoA and oxaloacetate condense in the first reaction of the TCA cycle to form citrate. Pyruvate and fatty acyl CoA can be converted to acetyl CoA. Pyruvate can also be converted to oxaloacetate by pyruvate carboxylase.

'3. List major metabolic intermediates synthesized from TCA cycle intermediates (add these to the diagram in #1).  '

Intermediates of the TCA cycle are used in the fasting state in the liver for production of glucose and in the fed state for the synthesis of fatty acids. They are also used to synthesize amino acids or to convert one amino acid to another. For gluconeogenesis: malate goes to make glucose (malate can be made from oxaloacetate). For fatty acids: citrate For amino acids: oxaloacetate is made from pyruvate, which by transamination forms aspartate and subsequently asparagines. Alpha ketoglutarate forms glutamate, glutamine, proline, and arginine.

'4. Describe briefly how the TCA cycle is regulated by substrate supply, allosteric effectors, covalent modification and protein synthesis.  '

The TCA cycle is regulated at several steps through citrate sythase, isocitrate dehydrogenase, and alpha ketoglutarate dehydrogenase activity. There is negative regulation or inhibition of all three enzymes when ATP, NADH, or products of the cycle are present in high levels. ADP, in indicating low energy levels, activates both citrate synthase and isocitrate dehydrogenase.

'5. List 4 common fates for the molecule pyruvate.  '

Pyruvate ==> lactate Pyruvate ==> acetyl CoA Pyruvate ==> oxaloacetate Pyruvate ==> alanine

'6. Explain why there is no synthesis of glucose from acetyl CoA, and know the fate of acetyl CoA. Understand the regulation of acetyl CoA production and why it is important.  '

Acetyl CoA cannot be used for gluconeogensis WHY? Therefore, acetyl CoA production is tightly regulated through regulation of pyruvate dehydrogenase. High levels of NADH and acetyl CoA directly inhibit pyruvate dehydrogenase. Further, high levels of NADH, acetyl CoA, and ATP indirectly inhibit the enzyme’s activity by stimulating the kinase that phosphorylates pyruvate dehydrogenase. In its phosphorylated form, it is inactive. ADP can inhibit this kinase, pyruvate dehydrogenase is dephosphorylated, and acetyl CoA is produced.

'7. Calculate the yield of ATP from the complete oxidation of glucose, pyruvate and acetyl CoA.  '

Glycolysis: 8 ATP equivalents 2 (Pryuvate ==> acetyl CoA) : 2 NADH or 6 ATP equivalents Each turn of the TCA: 3 NADH, 1 FADH2, 1 GTP or 12 ATP equivalents Net ATP from complete oxidation of glucose: 38 ATP

'8. Describe which intermediates accumulate during beri-beri deficiency and why.  '

Beri beri is a thiamine deficiency. Thiamine pyrophosphate is the prosthetic group of three important enzymes: pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and transketolase. Thus, beri beri results in an accumulation of pyruvate and alpha ketoglutarate.

'9. Describe what is meant by “insulin resistance” with regards to GLUT-4 receptors and diabetes.  '

Though insulin is still released in type 2 diabetes, and though that insulin binds to cell receptors, this binding does not result in the upregulation of the forward arm that recruits GLUT4 to the cell surface. Thus, glucose does not enter the cell readily.

'10. Know where the TCA cycle takes place, and why this location is important for linking it to energy production.  '

The TCA cycle takes place in the mitochondria, with all of the enzymes of the TCA cycle found in the mitochondrial matrix, with the exception of succinate dehydrogenase which is in the innermitochondrial membrane.