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Introduction to Metabolic Pathways; Glycolysis and TCA cycle


Glycolysis:'


1. Outline the sequence of reactions in aerobic and anaerobic glycolysis.


1. Glucose +ATP --> Glucose-6-phosphate (glucokinase in liver, hexokinase)**; #

2. --> Fructose-6-phosphate (phosphofructose isomerase)

3. + ATP' à Fructose-1,6-bisphosphate (phosphofructose kinase)** # 4. --> glyceraldehyde-3-phosphate (aldolase)

aldolase also converts F1,6BP to DHAP (dihydroxyacetone phosphate) DHAP à G3P (triose phosphate isomerase)

5. + Pi --> 1,3-bisphosphoglycerate + NADH (glyceraldehyde-3-phosphate dehydrogenase) 6. --> 3-phosphoglycerate + ATP (phosphoglycerate kinase) 7. --> 2-phosphoglycerate (phosphoglycerate mutase) 8. --> phosphoenol-pyruvate + H2O (enolase) 9. --> pyruvate + ATP (pyruvate kinase)** #

· Anaerobic glycolysis: pyruvate + NADH ßà L-lactate (lactate dehydrogenase) · **: hexokinase, phosphofructokinase (PFK-1), and pyruvate kinase are regulated

  1. irreversible



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).


· Steps that require ATP: o Glucose --> glucose-6-phosphate (hexokinase, glucokinase) o Fructose-6-phosphate -> Fructose-1,6-bisphophate (phosphofructokinase)


· Steps that yield energy: o Glyceraldehyde-3-phosphate -> 1,3 Biphosphoglycerate (yield NADH); G3P-dehydrogenase o 1,3 Biphosphoglycerate -> 3 phosphoglycerate (yields 1 ATP); phosphoglycerate kinase o phospho-enol pyruvate-> pyruvate (yield 1 ATP); pyruvate kinase


· Anaerobic glycolysis: requires NADH (pyruvate-> L-lactate)


· Net yield ATP for glycolysis: 2 NADH (3ATP) + 2ATP => 8 ATP


· Net yield for anaerobic glycolysis: 2ATP



3. Notate the net reaction for the conversion of glucose into pyruvate, & the # of ATP/NADH molecules formed.


· Glucose + Pi + 2NAD+ + 2ADP -> pyruvate + 2NADH + 2ATP + 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.


· Fed: both liver and brain go through glycolysis/TCA cycle; in fasting, liver converts glycogen/lactate/etc. to glucose, and glucose goes to brain; liver also converts fatty acids to ketone bodies and uses this as energy


· Major regulatory step: PFK-1 converts fructose-6-phosphate to fructose-1,6,-bisphosphate (using ATP) – irreversible


1. Allosteric inhibition by ATP: PFK-1 has both low & high affinity ATP binding sites; at low [ATP], ATP binds high affinity site, and at high [ATP], binds low affinity regulatory site (inhibition of activity)


2. Regulation by Fructose 2,6 bisphosphate · Phosphofructokinase converts fructose 6-phosphate to fructose 1,6 bisphosphate; fructose 1,6 bisphosphatase converts back to F6P · PFK-2 (phosphofrutokinase-2) and FB-2 (fructobisphosphatase) are two domains of same protein o PFK-2 converts F6P to fructose 2,6-bisphosphate; active when non-phosphorylated o FBP-2 converts fructose 2,6-bisphosphate back to F6P; active when phosphorylated o pKA phosphorylates PFK-2/FBP-2 so PFK-2 becomes inactive and FBP-2 is active · F2,6BP activates PFK1, inhibits FBP (so if enough F6P to create F2,6BP, then activates conversion of F6P to F1,6BP · Well fed: insulin inhibits pKA, so PFK-2 is active, so fructose-2,6-bisphosphate levels increase; F2,6BP activates PFK-1 and inhibits FBP, so fructose-6-phosphate is converted to fructose-1,6-bisphosphate · Fasting: glucagon activates pKA, so pFK-2 is inactive and FBP-2 is active. F2,6BP is converted back to F6P. so FBP is active, and PFK-1 is unstimulated. · F1,6BP stimulates pyruvate kinase. (feed-forward regulation)




5. Identify the step(s) in glycolysis:


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


· Phosphorylation of ADP to ATP is a thermodynamically unfavored process - it requires the input of energy to generate the high energy phosphate bond. The two steps in glycolysis where ATP is created drive this process by linked reactions involving the hydrolysis of another high energy phosphate bond in the glycolytic intermediate. (1,3 bisphosphoglycerate à 3 phosphoglycerate & PEP à pyruvate)


B. That exhibit feed-forward regulation.


· Fructose 1,6 bisphosphate (converted from fructose-6-phosphate by phosphofructokinase) is an activator of pyruvate kinase


· Pyruvate kinase converts phosphoenolpyruvate (PEP) to pyruvate


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


· 1,3 bisphosphoglycerate is converted to 3-phosphoglycerate by phosphoglycerate kinase (releasing ATP) o 1st step: 1,3 BPG à 2,3 bisphosphoglycerate by a mutase o 2,3 BPG is then converted to 3-PG by a phosphatase


· 2,3 BPG is high in red blood cells (trace in most cells) – it regulates binding of hemoglobin and oxygen


· 2,3 BPG binds to deoxy-Hb; reduces Hb affinity for O2 such that O2 is released in tissues


· Fetal hemoglobin has low affinity for 2,3 BPG, so has higher affinity for oxygen.


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


· Usually altered kinetics; 5-25% of normal activity, leading to decreased glycolysis


· Leads to hemolytic anemia in erythrocytes (no mitochondria so completely dependent on glycolysis)


· Alterations in membrane structural integrity, altered cell shape leads to short lived RBCs and anemia


· Most do not need any treatment, but severe cases can be treated with folic acid supplement or folic acid


· Pyruvate kinase: 95% of deficiencies; 4% are glucophosphate isomerase





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

Glucokinase

Where

In most tissues

Liver, b-cells

Km

Low (0.1mM), high glucose affinity

High (10mM), low glucose affinity

Vm

Low

High

Inhibition

Glucose 6-phosphate

None

Regulation

Inhibited by G6P

Inhibition by F6P, activation by glucose (sub-cellular localization)

· Phosphorylation of glucose by hexokinase and glucokinase is important because it traps the glucose into the cytosol, committing it to further metabolism. No carriers for G6P, and it is too polar to membrane diffusion.


· Regulation of glucokinase: when F6P is high, binds to glucokinase, promotes GKRP binding, and moves to nucleus; when F6P is high/glucose low, glucokinase dissociates from GKRP and binds glucose; active glucokinase then translocates to cytosol


· Glucokinase has low affinity and high rate, so when glucose levels are very high, glucokinase removes glucose quickly, minimizing hyperglycemia





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.


· Without sufficient energy production, there will be altered membrane structure, leading to altered cell shape and phagocytosis à so red blood cells are short lived


· Red blood cells get most of their energy from glycolysis (have no mitochondria)


· Without glycolysis, leads to hemolytic anemia





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


· Glucagon: decrease activity of glucokinase, PFK1, and pyruvate kinase.


· Insulin increases activity of glucokinase, PFK1, and pyruvate kinase.


· Glucagon/insulin also increases/decreases synthesis of glucokinase, PFK1, & pyruvate kinase (in liver).


· Insulin increases uptake of glucose (through increasing # of GLUT4 facilitated transporters)





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


· Pyruvate is converted to L-lactate by lactate dehydrogenase. High NADH production favors reduction to lactate.


· During intense exercise (and myocardial failure, hemorrhange, pulmonary embolism – cases with circulatory failure & decreased O2), lactate accumulates, causing decrease in pH – eventually diffuses into bloodstream; liver converts it back to pyruvate.


· Decreased ATP due to inability to carry out oxidative phosphorylation, but this may be enough to save life when circulation restored.


· Acidosis can cause cardiac arrhythmias, comas, death.


· Oxygen debt: amount of oxygen needed to remove lactic acid; often relates to patient morbidity/mortality


· Congenital lactic acidosis: most common is defect in pyruvate dehydrogenase; decreased ability to convert pyruvate to acetyl CoA o Brain is dependent on TCA cycle for most of energy o Severe – causes neonatal death; moderate: psychomotor defects, death in infancy; mild: occasional ataxia caused by carb-rich meal o No real treatment, but diet high in fat and low in carbs can help




10. Review the uptake of glucose into cells.


· In mucosal cells, co-transport with Na+ (secondary active transport)- SGLT-1.


· In most tissues, facilitated diffusion – driven by concentration gradient (Glut 1-5)


· Insulin increases GLUT4 transporters on membrane by stimulating vesicles containing GLUT4 to be recruited to the membrane.



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.


1. Oxaloacetate + acetyl coA-> citrate (citrate synthase)


2. Citrate -> isocitrate (aconitase)


3. Isocitrate -> a-ketoglutarate + NADH + CO2 (isocitrate dehydrogenase)


4. a-ketoglutarate -> uccinyl CoA + NADH + CO2 ('a'-ketoglutarate dehydrogenases)


5. succinyl CoA -> uccinate + GTP (succinate thiokinase)


6. succinate -> fumarate + FADH2 (succinate dehydrogenase)


7. fumarate-> L-malate (fumarase)


8. L-malate -> oxaloacetate + NADH (malate dehydogenase)




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


· Acetyl CoA: from glycolysis of glucose; pyruvate is converted to acetyl CoA by pyruvate dehydrogenase, releasing NADH


· Oxaloacetate: can be converted from pyruvate by pyruvate carboxylase (requires biotin) – is found in adipose tissue, brain, and liver (not muscle) & is activated by acetyl coA



3. List major metabolic intermediates synthesized from TCA cycle intermediates.


· TCA cycle intermediates used to create amino acids, glucose, fatty acids.


· Succinyl CoA can be used in biosynthesis of heme, can enter cycle from FA metabolism


· Citrate: used for fatty acid synthesis


· Amino acids – ex: pyruvate to alanine; also can form aspartate, glutamate, proline, glutamine, arginine





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


· Citrate synthase: (-): citrate, ATP, NADH, succinyl coA (+): ADP


· Isocitrate dehyrogenase: (-): ATP, NADH, Ca2+; (+): ADP


· a-ketoglutarate: (-): ATP, NADH, GTP, succinyl coA





5. List 4 common fates for the molecule pyruvate.


1. Converted to lactate by lactate dehydrogenase (requires NADH). 2. Converted to acetyl coA by pyruvate dehydrogenase (produces NADH). **only in mitochondria, not RBC 3. Converted to oxaloacetate by pyruvate decarboxylase. **only in mitochondria, not RBC 4. Converted to 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.


· Pyruvate is transported to the mitochondria matrix. Once it is converted to acetyl CoA, CoA does not allow it to leave the mitochondria.


· Pyruvate + CoA à acetyl CoA + NADH


· Pyruvate dehydrogenase complex is directly inhibited by NADH and acetyl CoA. Pyruvate dehydrogenase is also inactivated by kinase; the kinase is activated by ATP, NADH, and acetyl CoA. The kinase is inhibited by ADP. (So PDH is activated indirectly by ADP)



7. Calculate the yield of ATP from the complete oxidation of glucose, pyruvate and acetyl CoA. 38ATP




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


· Thiamine – B1, along with lipoic acid are co-factors for a-ketoglutarate dehydrogenase & pyruvate dehydrogenase


· So in beri-beri, aKGDH converts aKG to succinyl CoA; so aKG would accumulate.


· Pyruvate DH converts pyruvate to acetyl CoA. Pyruvate would accumulate à leading to accumulation of lactate. (also could be converted to acetyl CoA or oxaloacetate in mitochondria). Most likely, 2,3-bisphosphoglycerate would accumulate as well, if glucose was still taken in, F6P is converted into 2,3BPG.





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


· In hyperglycemia, insulin increases # of GLUT-4 receptors by stimulating vesicles to membrane. In insulin resistance, more insulin is needed to get the same cellular response. Diabetes mellitus type II occurs when the liver can no longer produce enough insulin to get the response needed.





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


· Occurs in mitochondria matrix; ETC is also in mitochondria (IMM).

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