Biochem SI 13- Glycolysis and Gluconeogenesis

Describe the two main stages of glycolysis and specify how many ATP are invested and generated in each.

Setup Phase→ Glucose is used to generate 2 GAP molecules→ 2 ATP consumed

Payoff Phase→ 2 GAP and 2 NAD+ are consumed to produce 2 NADH, 4 ATP, and 2 pyruvate

Net gain: 2 ATP and 2 NADH

What are the three irreversible reactions of glycolysis, and which enzymes catalyze them?

1→ Hexokinase

Glucose + ATP→ G-6P + ADP + H⁺

3→ PFK

F-6P + ATP→ F-1,6-BP + ADP + H⁺

10→ Pyruvate Kinase

2 PEP + ADP + H⁺→ 2 pyruvate + 2 ATP

Explain why the hexokinase reaction is considered “trapping” glucose within the cell

Because this reaction phosphorylates glucose using ATP and forms G-6P→ phosphate group is highly charged and cannot pass through the membrane

During glycolysis, which enzyme catalyzes the committed step and why is this step considered so important?

PFK→ it commits glucose to glycolysis because the product (F-1,6-BP) is only used in glycolysis

Identify the enzyme responsible for the only oxidation–reduction reaction in glycolysis and describe the products formed

Reaction 6: Glyceraldehyde-3-phosphate Dehydrogenase (GADH)

2 GAP + 2 NAD⁺ + 2 Pi→ 2 1,3-BPG + 2 NADH + 2 H⁺

Explain what is meant by “substrate-level phosphorylation” and list the reactions in glycolysis where it occurs

Direct transfer of a phosphate group from a high energy substrate→ ADP is phosphorylated to form ATP in 2 reactions:

Reaction 7 (Phosphoglycerate Kinase): 2 1,3-BPG + ADP + H⁺→ 2 3-phosphoglycerate + ATP

Reaction 10 (Pyruvate Kinase): 2 PEP + ADP + H⁺→ 2 pyruvate + 2 ATP

Possible because 1,3-BPG and PEP have higher phosphoryl transfer potential than ATP  

What is the overall net equation of glycolysis under aerobic conditions?

Glucose + 2 ADP + 2 NAD⁺ + 2 Pi→ 2 pyruvate + 2 ATP + 2 NADH + 4 H⁺

Define “energy charge” and explain how ATP and AMP levels regulate phosphofructokinase activity.

Energy charge→ cell is regulated by concentrations of AMP vs. ATP

High ATP/low AMP:

• ATP is a negative allosteric regulator of PFK→ binds to regulatory subunits and lowers its affinity for F-6P

• ATP is also allosteric inhibitor of pyruvate kinase→ ATP not produced at this step/PEP not converted to pyruvate

• PFK inhibition leads to a buildup of G-6P→ inhibits hexokinase (feedback inhibition)

• Inhibits glycolysis

Low ATP/high AMP:

• AMP is a positive allosteric regulator of PFK→ binds to regulatory subunits and raises its affinity for F-6P→ F-1,6-BP produced

• F-1,6-BP is an allosteric activator of pyruvate kinase (feed forward stimulation)

• G-6P no longer accumulates→ hexokinase is active

• Stimulates glycolysis

A muscle cell has high ATP and low AMP concentrations. Predict how this would affect phosphofructokinase activity and glycolytic flux.

This will inhibit PFK activity and slow glycolytic flux→ ATP is neg, allosteric regulator of PFK and Pyruvate Kinase/G-6P buildup inhibits hexokinase

Contrast the regulation of glycolysis in muscle versus liver. What is each tissue primarily using glycolysis for?

Muscle:

• Energy charge ([ATP] vs. [AMP])

• Uses glycolysis for energy production during exercise

Liver:

• Hormonal regulation

1. Insulin→ high BG→ glycolysis

2. Glucagon→ low BG→ gluconeogenesis

• Balance between blood glucose and glucagon

• Glycolysis provides carbon skeletons for biosynthesis

Why does gluconeogenesis require four unique enzymes instead of simply reversing all glycolytic reactions?

Because 3 reactions in glycolysis are irreversible (negative delta G):

2 needed to reverse reaction 10, 1 for reaction 3, 1 for reaction 1

Describe the role of pyruvate carboxylase in gluconeogenesis, including its location, cofactor, and energetic requirements.

Pyruvate carboxylase:

• Located in mitochondrial matrix

• Uses biotin cofactor/prosthetic group that carries activated CO₂ for carboxylation of pyruvate→ oxaloacetate

• Uses 2 molecules of ATP and HCO₃^-

Explain how pyruvate is converted into phosphoenolpyruvate (PEP) during gluconeogenesis, and why this step must occur in two reactions.

It is carboxylated to oxaloacetate using pyruvate carboxylase (uses 2 ATP) and decarboxylated/phosphorylated by PEP carboxykinase (uses 2 GTP)→ happens in two steps to compensate for energetic cost of phosphorylating pyruvate directly and bypasses irreversible pyruvate kinase step

Compare the reactions catalyzed by phosphofructokinase and fructose-1,6- bisphosphatase. How do these enzymes coordinate reciprocal regulation?

PFK→ glycolysis→ converts F-6P to F-1,6-BP

FBPase→ gluconeogenesis→ converts F-1,6-BP to F-6P

When one is active, the other is inactive→ prevents futile cycling/waste of energy

Gluconeogenesis consumes six high-energy phosphates per glucose molecule synthesized. Why is this energetically favorable even though it costs more energy than glycolysis produces?

Because maintaining blood glucose and brain fuel supply outweighs energy cost; coupling with ATP hydrolysis makes the pathway thermodynamically favorable under cellular conditions.