Case 4 BIOCHEM: Metabolism, glycolysis, CAC, gluconeogenesis

Question 1

The purpose of metabolism

Answer 1

• oxidation of food to provide energy
• conversion of food molecules to new cellular material & essential components of the body
• processing and excretion of waste products

Question 2

Define ‘intermediary metabolism’

Answer 2

All reactions that store and extract chemical energy from nutrient molecules, and synthesise low molecular-weight compounds and energy-storaged compounds

Question 3

List the main dietary carbohydrates and their sources

Answer 3

1. Amylose (linear starch) - potatoes, rice, starch
2. Amylopectin (branched starch) - potatoes, rice, starch
3. Sucrose (disaccharide) - deserts, sweets, sugar
4. Lactose (disaccharide) - milk
5. Fructose (monosaccharide) - fruits, honey
6. Glucose (monosaccharide) - fruits, honey

Question 4

Outline how dietary carbohydrates are absorbed by the gut

Answer 4

1. lactose - broken down into galactose + glucose which are transported across the mucosal membrane into the bloodstream
2. starch - broken down into glucose molecules & absorbed similarly
3. glucose - directly absorbed
4. sucrose - broken down into fructose and glucose

Question 5

Why can’t the products of muscle glycogenolysis supply energy to the rest of the body?

Answer 5

Glycogen is broken down into Glucose-6-Phosphate, which is negatively charged, making the membrane impermeable to it. Muscles lack the enzyme that converts G6P to glucose, therefore cannot release free glucose into the blood.

Question 6

How is dietary fat stored?

Answer 6

In the form of triacylglycerol in the adipose tissue

Question 7

Outline the forms of energy supply to the brain

Answer 7

• normal conditions: glucose

- starvation: ketone bodies produced by the liver (ketogenesis)

Question 8

The role of ATP in metabolism

Answer 8

ATP links energy-releasing processes to energy-requiring processes in the cell, such as muscle contraction, active transport, biosynthesis, and signal transduction.

Question 9

How is ATP synthesised?

Answer 9

ATP generation from ADP is endergonic, thus the process must be coupled to thermodynamically favourable processes (exergonic) such as substrate level phosphorylation (oxidation of fuel molecules from food).

Question 10

The significance of ATP hydrolysis having a high activation energy

Answer 10

This means that ATP hydrolysis requires catalysis, ensuring that energy is not being squandered.

Question 11

The significance of glucose metabolism in energy production

Answer 11

• glucose catabolism is the converging point of amino acid and fatty acid metabolism
• metabolism of other monosaccharides (fructose and galactose) produce metabolites that enter the glucose catabolic pathway

Question 12

Outline the stages of glucose oxidation

Answer 12

• Glycolysis produces pyruvate, NADH and ATP
• pyruvate oxidation produces acetyl coA
• acetyl coA is fed into the citric acid cycle, producing NADH, FADH2 and GTP
• NADH and FADH2 are oxidatively phosphorylated by the electron transport chain to produce ATP

Question 13

The general functions of glycolysis

Answer 13

• In glycolysis, glucose is oxidised to pyruvate. This process serves two main functions:
  1. Generate energy in the form of ATP
  2. Provide carbon skeletons for biosynthesis of other compounds

Question 14

Outline the characteristics of the 2 phases of glycolysis

Answer 14

• in the first phase, a series of five reactions break down glucose to two molecules of glyceraldehyde-3-phosphate. In the second phase, five subsequent reactions convert these two molecules into two molecules of pyruvate.
• Phase 1 consumes 2 ATP molecules, the later stages result in the production of 4 molecules of ATP.

Question 15

Explain the regulation of PFK and its role in glucose catabolism

Answer 15

• PFK (phosphofructokinase) is the ‘valve’ controlling the rate of glycolysis.
• this enzyme has 2 substrates: ATP and F-6-P
• In addition of its role of being a substrate, ATP is also an allosteric inhibitor of this enzyme. Thus PFK has two distinct binding sites for ATP: a high-affinity substrate site and low-affinity regulatory site. In the presence of high ATP concentrations, PFK behaves cooperatively and increases the Km of F-6-P.
• AMP reverses the inhibition due to ATP. AMP levels in cells rise when ATP is being hydrolysed.
• essentially, the activity of PFK increases when the energy status falls and is decreased when the energy status is high. The rate of glycolytic activity decreases under high concentrations of ATP and increases under low concentrations of ATP and high levels of AMP.
• glycolysis and CAC are coupled via PFK, because citrate (an intermediate of CAC) is an allosteric inhibitor of PFK. Therefore when CAC reaches saturation, glycolysis slows down.
• note: AMP regulation on PFK is NOT cooperative (hyperbolic curve)

Question 16

Define ‘substrate-level phosphrylation’

Answer 16

the synthesis of ATP from the breakdown of high-energy compounds (BPG or PEP)

Question 17

Explain how energy is released through the oxidation of G3P, and how it is used to generate BPG

Answer 17

• oxidation of G3P is highly exergonic

G3P + NAD + Pi -> 1,3-BPG + NADH

• some of the energy is trapped in NADH (as the electro carrier)
• the remaining energy is used to phosphorylate the oxidised G3P at carbon 1 to generate BPG

Question 18

Outline the biosynthetic intermediates in glycolysis

Answer 18

1. G-6-P - can be used to produce glycogen
2. other glycolysis intermediates - amino acids
3. 3-phosphoglycerate - 2,3-BPG
4. Dihydroxyacetone phosphate - glycerol-3-p for fat synthesis

Question 19

Outline how fructose and galactose metabolism converge onto glycolysis

Answer 19

1. fructose: metabolised into 2 trios phosphates

2. galactose: can be metabolised into G-1-P, which is converted to G-6-P

Question 20

Outline the reoxidation of NADH under aerobic vs. anaerobic conditions

Answer 20

1. under aerobic conditions: NADH is reoxidized via mitochondrial electron transport chain
2. under anaerobic conditions: NADH is reoxidized via the reduction of pyruvate into lactate

Question 21

Outline the functions of CAC

Answer 21

1. generation of energy in the form of GTP
2. providing biosynthetic precursors
   • citrate -> fatty acids
   • oxaloacetate -> glucose via gluconeogenesis
   • succinyl coA -> haem

Question 22

Identify the 3 sources of Acetyl CoA

Answer 22

1. amino acids
2. glucose
3. fatty acids

Question 23

Identify the enzyme that oxidises pyruvate and its significance in CAC

Answer 23

• pyruvate dehydrogenase (PDH)
• this process is highly exergonic: some energy trapped in NADH and others stored in acetyl CoA
• irreversible thus functions as major control point

Question 24

Explain the role of CAC in energy extraction

Answer 24

• fatty acids and glucose are oxidised to acetyl coA, a CAC substrate.
• CAC completes the oxidation of glucose or fatty acids to CO2 via acetyl coA oxidation
• oxidation of acetyl CoA to CO2 = highly exergonic, and some of the energy is trapped in NADH and FADH2, and the remaining energy is used in substrate-level phosphorylation to form GTP.

Question 25

Define ‘anaplerotic reactions’ and explain their role in the CAC

Answer 25

• the cell also feeds back into the CAC cycle through other reactions* to replenish the intermediates used in biosynthetic processes
• anaplerotic reactions: ‘filling up’ reactions
• pyruvate carboxylase and amino acid catablism serve as anaplerotic reactions of CAC
• pyruvate carboxylase = most important of the anaplerotic reactions. It exists within the mitochondria of animal cells and provides a direct link between glycolysis and the CAC.
  Pyruvate carboxylase has an absolute allosteric requirement for acetyl-coA, thus when acetyl-CoA levels exceed the oxaloacetate supply (indicating a depletion of OAA), allosteric activation of pyruvate carboxylase converts acetyl CoA into OAA to allow the CAC to continue.
• the catabolism of amino acids produce pyruvate, acetyl CoA, oxaloacetate, fumarate, and succinate. All of which may be oxidised by the CAC.

Question 26

Explain how the CAC is regulated via pyruvate dehydrogenase

Answer 26

• Because the dehydrogenation of pyruvate is irreversible, the conversion of it to acetyl-CoA commits it to CAC.
• Pyruvate dehydrogenase (PDH) is under product inhibition and is further regulated by phosphorylation and dephosphorylation of the enzyme itself.
• High levels of either product, acetyl coA or NADH, allosterically inhibit the PDH enzyme.
• PDH is also sensitive to the energy status of the cell. High ATP/ADP, Acetyl-CoA/CoA, NADH/NAD levels causes the phosphorylation of the enzyme, which inhibits it.
• high levels of NAD+ and CoA indicate a depletion of Acetyl CoA and NADH, which activate the enzyme

Question 27

Explain the Cori Cycle

Answer 27

Glucose produced by gluconeogenesis in the liver is released into the blood and is subsequently absorbed by the brain, heart, muscle and red blood cells to meet their metabolic needs. In turn, pyruvate and lactate produced in these tissues are returned to the liver to be used as gluconeogenic substrates.

Question 28

Explain how gluconeogenesis and glycolysis are reciprocally regulated and why this is significant

Answer 28

The processes of gluconeogenesis and glycolysis must be reciprocally regulated so that when glycolysis is active, gluconeogenesis is inhibited, and vice versa. These limitations are overcome by having unique reactions within the routes of glycolysis and gluconeogenesis (by having unique enzymes), rather than having completely shared pathway.

Question 29

Outline the process of gluconeogenesis in relation to glycolysis

Answer 29

“Something borrowed, something new”

• Gluconeogenesis employs four different reactions, catalysed by four different enzymes, for the 3 irreversible steps in glycolysis (highly exergonic steps). Essentially, seven of the 10 steps of glycolysis are merely reversed in gluconeogenesis.

HOW THE LIVER BYPASSES THE 3 IRREVERSIBLE STEPS OF GLYCOLYSIS

• The conversion of pyruvate to phosphoenolpyruvate (PEP) initiates gluconeogenesis. This process is accomplished by two unique reactions: pyruvate carboxylase converts pyruvate to oxaloacetate; then PEP carboxylase catalyses the conversion of oxaloacetate to PEP.
• Conversion of F-1,6-P to F-6-P is catalysed by fructose-1,6-phosphatase.
• The final step to producing glucose, the hydrolysis of G-6-P is mediated by glucose-6-phosphatase .

Question 30

Explain the conversion of pyruvate to PEP: how it is regulated and compartmentalised

Answer 30

CONTROL:
- pyruvate carboxylase is allosterically activated by Acetyl-CoA. Acetyl-CoA is the prime substrate for CAC cycle, and OAA is an important intermediate in both CAC and gluconeogenesis. If levels of ATP and/or acetyl-CoA are low, pyruvate is directed into the CAC, which promotes the synthesis of ATP. If NADH and Acetyl-CoA levels are high (from B oxidation), pyruvate oxidation is inhibited and pyruvate carboxylase is activated, converting pyruvate to OAA which is consumed in gluconeogenesis.

COMPARTMENTALISATION:
- pyruvate carboxylase is only found in the mitrochondria, whereas the next enzyme in gluconeogenesis, PEP carboxykinase, is found in both mitochondria and cytosol. Pyruvate is transported into the mitochondrial matrix, where it can either be converted into acetyl coA or oxaloacetate by pyruvate carboxylase. Oxaloacetate cannot be transported directly across the membrane, therefore it is converted to malate. Cytosolic malate is converted back to oxaloacetate for the continuation of gluconeogenesis.

Question 31

Outline the control of the conversion of Fructose-1,6-bisP to Fructose-6-P.

Answer 31

• enzyme: fructose-1,6-bisPhosphatase

- inhibited by F-2,6-B,P and high levels of AMP