Case 8: Pyruvate Oxidation and Citric Acid Cycle

What’s the overall goal of the pyruvate dehydrogenase complex (PDH)?

The overall goal of the PDH complex is to form acetyl-CoA from pyruvate (made by glycolysis) through pyruvate decarboxylation.

Under anaerobic conditions, what is the fate of pyruvate in human skeletal muscle?

Under anaerobic conditions in human skeletal muscle, pyruvate is converted to lactate with lactate dehydrogenase. The reaction oxidizes NADH to NAD+, allowing glycolysis to keep producing energy

Under anaerobic conditions, what is the fate of pyruvate in yeast? 

Under anaerobic conditions, pyruvate is converted to acetaldehyde with pyruvate decarboxylase. The acetaldehyde is then converted to ethanol with alcohol dehydrogenase. The reaction oxidizes NADH to NAD+, allowing glycolysis to keep producing energy.

What effects do the following conditions have on PDH complex?

• low energy charge

• phosphorylation of PDH

• high levels of acetyl-CoA

• high levels of NADH

• low energy charge = activate the PDH complex

• phosphorylation of PDH = inhibit the PDH complex.

• high levels of acetyl-CoA = inhibit the PDH complex (binds to E2)

• high levels of NADH = inhibit the PDH complex (binds to E3)

Predict the effect of a mutation on the PDH kinase. 

PDH kinase phosphorylates the PDH to inhibit the complex. A mutation on the PDH kinase would cause the PDH to remain active, meaning pyruvate continues to convert to acetyl-CoA. This would cause a higher demand for pyruvate, making the cell use more glucose and lowering blood sugar levels.

Inhibitors of PDH kinase have been proposed as potential treatments for type 2 diabetes. Suggest a biochemical rationale for this proposal. 

PDH kinase phosphorylates PDH to inhibit the complex. Inhibiting PDH kinase would result in pyruvate continuously converting to acetyl-CoA, increasing the demand for pyruvate. This would cause the cell to use more glucose to make pyruvate, therefore taking the glucose from the bloodstream and lowering blood glucose levels.

In type 2 diabetes, patients have insulin resistance, meaning the insulin can’t inhibit gluconeogenesis (keeps making glucose). Keeping the PDH complex active can reduce the amount of blood glucose, therefore lowering symptoms of the disease.  

What’s the overall goals of the Citric Acid Cycle?

There overall goals of the citric acid cycle is to…

• generate NADH and FADH2: provides electrons for oxidative phosphorylation.

• generate ATP

• generate biosynthetic precursors that are used to make amino acids and other molecules

What is the net equation of the citric acid cycle, per glucose molecule?

2 acetyl-CoA + 6 NAD⁺ + FAD⁺ + 2 ADP + 2 Pi + 4 H2O → 2 CoA + 4 CO2 + 6 NADH + 2 FADH₂ + 2 ATP + 4 H⁺

How is oxaloacetate replenished? Under what cellular conditions would this enzymatic step be favored?

Oxaloacetate is replenished by carboxylation of pyruvate (catalyzed by pyruvate carboxylase). This is favored when there’s high levels of acetyl-CoA (to activate pyruvate carboxylase) and a low energy charge (which directs oxaloacetate to the CAC).

Fats are converted to acetyl CoA and then further processed through the citric acid cycle. We learned that glucose could be made from oxaloacetate. Why do we not simply convert fats into glucose? 

• glucose can be made from oxaloacetate (gluconeogenesis)

• fats can be converted to 1 acetyl-CoA to then go to CAC (which makes oxaloacetate)

• however, when trying to make oxaloacetate from fats, for every 2 carbon acetyl-CoA, 2 carbon is lost as CO2 before oxaloacetate can even be made.

Therefore, the fats cannot be converted to glucose because the carbon from acetyl-CoA from fats is used up and lost as CO2 in the CAC before oxaloacetate can even be made.

list the key regulatory steps of the citric acid cycle and describe how they are regulated.

Step 1: Citrate Synthase

• acetyl-CoA and oxaloacetate produce citrate and CoA.

• This reaction is inhibited by high-energy signals (ATP, NADH, citrate)

• activated by ADP (signals low energy)

• When oxaloacetate binds to citrate synthase, it induces a conformational change, allowing the binding of acetyl-CoA.

Step 3: Isocitrate Dehydrogenase

• isocitrate and NAD+ produce α-ketoglutarate, producing NADH and releasing CO₂.

• inhibited by NADH (indicates sufficient energy)

• activated by ADP

• when energy levels are high, the activity of the enzyme decreases

Step 4: α-Ketoglutarate Dehydrogenase Complex

• α-ketoglutarate and NAD+ and CoA produce succinyl-CoA, producing NADH and releasing CO₂.

• inhibited by high NADH levels (indicating enough energy)

• activated by calcium ions (Ca²⁺), which signal the need for more ATP

When 1 mole of Glucose is oxidized via glycolysis and Citric acid cycle, how many moles of ATPs, NADH, FADH2 and CO2 are formed?

ATP = 4

Glycolysis

• hexokinase: -1

• phosphofructokinase: -1

• phosphoglycerate kinase: +2

• pyruvate kinase: +2

CAC

• succinyl-CoA synthetase: +2

6-2 = 4

NADH = 10

Glycolysis

• glyceraldehyde 3-phosphate dehydrogenase: +2

PDH Complex: +2

CAC

• isocitrate dehydrogenase: +2

• a-ketoglutarate dehydrogenase complex: +2

• malate dehydrogenase: +2

FADH2 = 2

CAC

• succinate dehydrogenase: +2

CO2 = 6

PDH Complex: +2

CAC

• isocitrate dehydrogenase: +2

• a-ketoglutarate dehydrogenase complex: +2

List three substances that lower the pH of blood

• lactate

• ketone bodies

• CO2

Why might a person have elevated concentrations of pyruvate and lactate in the blood?

• When the PDH complex isn’t working, pyruvate cannot be converted to acetyl-CoA.

• Therefore, pyruvate accumulates in the cytoplasm. It is then converted to lactate by lactate dehydrogenase to regenerate NAD⁺, which is needed to continue glycolysis.

The high concentration of α-ketoglutarate could be due to the loss or inactivation of which enzyme?

inactivation of α-ketoglutarate complex

What other cofactors or co-substrates does the αketoglutarate dehydrogenase complex require to function?

• TPP, lipoic acid, FAD

• CoA and NAD+

The man in this case appears to have experienced simultaneous loss of activity of the PDH complex and the αketoglutarate dehydrogenase complex.

What would be the mostly likely cause of this simultaneous loss of enzymatic activity in this case?

TPP is a shared cofactor between the two complexes. The simultaneous loss of enzymatic activity is most likely due to TPP deficiency. 

Describe the most likely explanation of these test results. (why is there low pH of blood)

The lack of TPP in the E1 subunit of the PDH complex would have prevented it from converting pyruvate into acetyl-CoA and converting to lactate instead. This build up of lactate would have decreased the blood pH of the man.  

Based on what you know about this case, which of the three catalytic subunits of the αketoglutarate dehydrogenase complex would you expect to demonstrate decreased activity in this subject? 

The E1 subunit would demonstrate decreased activity due to it containing TPP.  

Based on all the evidence you have gathered in this case, what is the most likely underlying cause of this man's heart failure and death?

The underlying cause of the man’s heart failure and death is most likely due to the lack of TPP because of the vitamin B1 deficiency. The lack of TPP would have impaired PDH complex and it would not have been able to convert pyruvate into acetyl-CoA.

The build up of pyruvate would lead to a build up of lactate, therefore lowering the pH. This low pH would cause cardiac cell degradation, leading to the heart attack.   

What is Beriberi? How is Wernicke-Korsakoff syndrome related to Beriberi? What is the cause of this suite of diseases?

The Beriberi disease is caused by a deficiency of vitamin B1. Without thiamine, TPP is unable to be activated, impairing the E1 subunit, and leading to a non-functional PDH complex. There are two types, dry and wet Beriberi. Dry Beriberi impacts the nervous system and causes weakness. Wet Beriberi impacts the heart and circulatory system. The Wenicke-Korsakoff syndrome is related to the Beriberi disease. It is also due to vitamin B1 deficiency and is often found in alcoholics. The Wenicke-Korsakoff syndrome consists of psychiatric symptoms due to the deficiency. The brain mainly uses glucose to make acetyl-CoA, but the lack of thiamine disables the citric acid cycle. This leads to not enough energy being produced, along with lactate buildup. The lack of energy in the brain results in the decreased production of neurotransmitters such as acetylcholine and GABA. Symptoms include confusion and loss of mental activity.

The high alanine level is most likely due to ____ .

The high glutamate level is most likely due to ____

high pyruvate level, high a-ketoglutarate level