Carbohydrates- glycolysis (Part 2)

Question 1

What are the three enzymes that regulate glycolysis? What are the differnt types of regualtion seen in glycolysis?

Answer 1

Regulated at three steps (irreversible reactions)

1. Hexokinase/glucokinase
2. Phosphofructokinase-1 (PFK-1)
3. Pyruvate kinase

Different types of regulation seen in glycolysis

• Allosteric mechanism
• Covalent modification (phosphorylation/Dephosphorylation)
• Induction/repression

Question 2

How are hexokinase and glucokinase regulated via induction and repression by enzymes/ products?

Answer 2

Hexokinase
•Hexokinase is allosterically inhibited by its product, glucose 6-phosphate

Glucokinase

• Indirectly inhibited by fructose 6-phosphate and indirectly activated by glucose – mediated by glucokinase regulatory protein (GKRP) in the liver
• Induced by insulin; repressed by glucagon

Question 3

•Phosphofrustokinase-1 is one of the major regulatory enzymes of glycolysis. What is PFK-1 inhibited by and what is PFK-1 activated by? What is the most potent activator of PFK-1?

Answer 3

Allosteric regulation of PFK-1

• *•PFK-1 inhibited by high levels of**
• >ATP (signals ‘energy-rich’ state)
• >citrate (build up in TCA cycle)
• *•PFK-1 activated by high levels of**
• >AMP (signals ‘low energy’ status)
• >Fructose 2,6-bisphosphate; the most potent activator of PFK-1; activates PFK-1 even when ATP levels are high

Question 4

How is Fr 2,6 bisphosphate made and by which enzyme? What coverts Fr, 2 ,6 Phosphate back to Fr 6-P? PFK-1 and FBP-2 are both a _______? How are these enzymes regulated?

Answer 4

• Fructose 6-P is converted to Fr 2,6-bisphosphate by PFK-2
• Fr 2,6-BP is converted back to Fr 6-P by fructose 2,6-bisphosphatase (FBP-2)
• PFK-2, FBP-2; two enzymatic activities of a ‘bi-functional’ protein
• When kinase is active; phosphatase is inactive
• The bi-functional protein is regulated by phosphorylation/ dephosphorylation (covalent modification) mediated by hormones

Question 5

Explain the regulation of PFK2/FBP-2 in the liver by covalent modification

Answer 5

•PFK-2/FBP-2, a bifunctional protein with two enzyme activities. Regulated by phosphorylation and Dephosphorylation. In the liver, the kinase is active in the dephosphorylated form whereas the phosphatase is active in the phosphorylated state.

In well feed state -> high glucose levels -> inc insulin -> activates PFK2-> inc fructose 2,6 phosphate-> activated PFK-1-> inc glycolysis

During starvation-> low blood glucose-> inc glycagen-> activated FBP-2 -> dec fructose 2,6 bisphosphate-> no activation of PFK-1-> dec glycolysis

Question 6

How is pyruvate kinase regulated in the liver?

Answer 6

Regulation of pyruvate kinase (PK) in liver

• Allosterically activated by fructose 1,6-bisphosphate (feed-forward activation)
• Allosterically inhibited by ATP

Covalent modification

• Pyruvate kinase regulated through covalent modification
• Active in the dephosphorylated state and inactive in the phosphorylated state

Question 7

What is the significance of glycolysis?

Answer 7

1.Provides energy
2.Generates 2,3-BPG (Rapoport-Leubering shunt)
•2,3-BPG present in high concentration in RBCs
•lowers oxygen affinity of hemoglobin
•Helps in unloading of oxygen from hemoglobin in tissues

3. Provides glycerol 3-phosphate for synthesis of triacylglycerol (Fat)

4. Provides precursors for non-essential amino acid synthesis (pyruvate à alanine which is a non essentail amino acid )

Question 8

What are the three enzymes that insulin induces and glycagon represses?

Answer 8

Regulation of glycolytic enzymes by induction & repression

• Insulin induces the three key enzymes (glucokinase, PFK-1, PK)
• Glucagon represses the same three key enzymes

Question 9

What are the clinical agents that inhibit glycolysis?

Answer 9

->Iodoacetamide, N-ethylmaleimide (thiol reagents, react with –SH group in active site) inhibit glyceraldehyde 3-phosphate dehydrogenase
->Fluoride inhibits enolase; Fluoride coated tubes used to collect blood for glucose estimation/ testing
->Arsenate prevents net ATP production by glycolysis
•Pentavalent arsenic (arsenate) competes with Pi as a substrate for G3P Dehydrogenase
•Allows glycolysis to continue without ATP generation

Question 10

Clinical correlation: Pyruvate kinases deficiency. What happens when there is a pyruvate kinase deficieny? what type of anemia does it lead to? What increases in this deficieny? What are the features of this deficiency?

Answer 10

•Generally restricted to RBC
dec in rate of glycolysis, depletion of ATP Atp is needed to form membrane pump->Loss of membrane integrity, loss of biconcave shape
•Such RBCs are destroyed by macrophages in spleen
•Chronic hemolytic anemia, normochromic & normocytic
•One of the common causes of hemolytic anemia due to enzyme deficiency
•↑in 2,3 BPG, resulting in ↓ed affinity of O2 for Hb ( because of hemolysis)
•Features: Pallor, fatigue, jaundice, enlargement of spleen, shortness of breath, gallstones (cholecystolithiasis), ↓ed Hb level, **Absence of Heinz bodies **
•
•Treatment: May require regular blood transfusion