Glycolysis & the TCA Cycle

Glycolysis
Glycolysis is the anaerobic process that occurs in the cytosol by which glucose is broken down into
pyruvate. A small amount of ATP is generated during this process by substrate-level
phosphorylation. Reducing equivalents (NADH and FADH2) will be used to later convert their redox
potential to phosphate potential for the production of ATP.

1. Glycolysis occurs in 10 steps beginning with glucose and ending with pyruvate. Complete the
diagram below by identifying the substrate/product, enzyme or bi-product for each step.
2. Glycolysis can be broken down into two distinct phases: Energy Investment and Energy
Payoff. Which steps (#) fall into each category? What products are consumed and produced
during the investment and payoff phases. What is the net ATP generation?

3. a. Based on the graph below, which steps are the key regulatory steps of glycolysis and what
is the enzyme that catalyzes each?
Cumulative Free Energy

b. Why are these three steps key to maintaining flux from glucose to pyruvate?

c. Are these steps likely reversible or irreversible?

d. Which enzyme is considered the rate-limiting step of glycolysis?

4. Regulation of glycolysis occurs primarily by allosteric modulation of the activity of these key
regulatory enzymes. Below is a table with positive and negative allosteric modulators of the
three regulatory enzymes for glycolysis.

Identify the enzyme in each row and label modulators on glycolysis figure.

Enzyme Positive Modulators Negative Modulators

Glucose 6-phosphate
AMP, ADP,
ATP, NADH, Citrate
Fructose 2,6 bisphosphate
Fructose 1,6 Bisphosphate ATP, Acetyl-CoA

5. Does allosteric modulation change the enzyme activity or enzyme concentration?

6. How do allosteric modulators change the Km and Vmax of an enzyme?

7. What does this mean for the overall flux of substrates through glycolysis? Why is ATP a
negative allosteric modulator at many of the key regulatory sites?
TCA Cycle
Also known as the Krebs cycle or citric acid cycle, this aerobic process occurs in the
mitochondrial matrix primarily generating reducing equivalents, which are utilized by the
electron transport chain for ATP production. The cycle begins with Acetyl-CoA, which can be
derived from many sources including carbohydrates, fatty acids, and amino acids.

1. How do we get Acetyl-CoA from glycolysis? List the enzyme, reactants, and products.

2. Add the above step to the figure below and complete the diagram by identifying the
substrate/product, enzyme or bi-product for each step of the TCA cycle. Label where each
CO2, ATP equivalent, NADH and FADH2 is produced.
3. What step changes from a 6-carbon structure to 5 carbons? 5 Carbons to 4?

4. Based on the graph below, what are the key regulatory steps in the TCA
cycle? List the substrate, enzyme and products of these steps.
Which enzyme is considered the rate-limiting step?

Steps of the TCA

0 1 2 3 4 5 6 7 8
0
-20
Cumulative Free Energy

-40
-60
-80
-100
-120
-140

5. Allosteric modulation of enzymes is also one of the primary modulators of flux through
the TCA cycle. Below is a table with positive and negative allosteric modulators of the
three regulatory enzymes for the TCA cycle.
Identify the enzyme in each row and draw the modulators on the TCA figure.

Enzyme Positive Modulators Negative Modulators

NADH, Citrate, ATP,
ADP
Succinyl-CoA
ADP, Ca2+ NADH, ATP

ADP, Ca2+ NADH, Succinyl-CoA, ATP

6. Adenylate charge is a measure of the energy status of cells and incorporates ATP, ADP, and
AMP into a ratio. A typical cellular value is around 0.80 and metabolic reactions can be
controlled by changes in energy status.
What does a high adenylate charge do to the flux of substrates through the TCA
cycle? How is this action carried out through enzyme regulation?

[𝐴𝑇𝑃] + 0.5[𝐴𝐷𝑃]
𝐴𝑑𝑒𝑛𝑦𝑙𝑎𝑡𝑒 𝐶ℎ𝑎𝑟𝑔𝑒 =
[𝐴𝑇𝑃] + [𝐴𝐷𝑃] + [𝐴𝑀𝑃]