Regulation of Glycolysis

Question 1

What does Glycolysis convert?

Answer 1

One molecule of glucose into two molecules of pyruvate with the generation of two molecules of ATP

Question 2

Where does Glycolysis occur?

Answer 2

• Glycolysis occurs in all tissues, particularly important for energy in brain and Rbc’s and also in contracting skeletal muscle
• Rbc’s account for 10%
  of the total bodies usage

Question 3

What are the two stages that Glycolysis can occur in ?

Answer 3

• Stage 1 = traps glucose in the cell and modifies it so that it can be cleaved into a pair of
  phosphorylated 3-carbon compounds
• Stage 2 = oxidises the 3-carbon compounds to pyruvate while generating two molecules of ATP

Question 4

In the glycolytic pathway, metabolised glucose produces?

Answer 4

• ATP by substrate level and oxidative
• Glycerol-3-phosphate for fat synthesis
• Acetyl CoA for fat and cholestrol synthesis
• Amino acids

Question 5

What is Fructose 1,6-bisphosphate generated from? (committed step of glycolysis)

Answer 5

Glucose 6-phosphate

Question 6

Explain a little about the generation of Fructose 1,6-bisphosphate ?

Answer 6

The carbohydrate is trapped in the fructose form by the addition of a second phosphate to form fructose 1,-6 bisphosphate. This irreversible reaction is catalysed by the allosteric enzyme phosphofructokinase (PFK)

Question 7

Major point of control lies at committed step of glycolysis, catalysed by ?

Answer 7

PFK1, a heterotetrameric enzyme: fructose-6-phosphate (F6P) and ATP are the substrates

Question 8

What kind of kinetics does PK1 show ?

Answer 8

PFK1 shows sigmoidal kinetics: allosteric regulators bind at the interface between subunits of
the enzyme, shifting it between T and R states

Question 9

What stimulates PKF1 and glycolysis ? and what inhibits it?

Answer 9

• Low energy charge (AMP, ADP) stimulates

- High energy (ATP) inhibits it

Question 10

What are the other key regulators that inhibit and stimulate ?

Answer 10

Note other key regulators citrate (inhibits: signal of fatty acid oxidation) and fructose-2,6-bisphosphate (a pure regulator: activates)

Question 11

What regulates the committed step of glycolysis?

Answer 11

Reciprocally regulated with fructose-1,6-bisphosphatase, which catalyses the reverse reaction (you lose a high energy phosphate to do this)

Question 12

Purpose of Fructose 2,6 bisphosphate ?

Answer 12

Not involved in metabolic pathways, just a regulator: another product from F6P that acts solely to reinforce allosteric control on PFK-1

Question 13

What does Fructose 2,6 bisphosphate activate and inhibit ?

Answer 13

• Activates glycolysis: strongest allosteric activator of PFK1
• Inhibits gluconeogenesis: strong inhibitor of fructose-1,6-bisphosphatase

Question 14

What activates and inhibits PFK2 ?

Answer 14

Activated by:

1. F-6-P
2. AMP
   - increases glucose concentration, glycogen breakdown (muscle) and contraction (low ATP)

Inhibited by:

1. Citrate
   - increases fatty acid oxidation (TCA cycle overload)

Question 15

Explain the reciprocal control of committed step by

Fructose-2,6-bisphosphate?

Answer 15

• Glycolysis is accelerated by increased Fru-2,6BP by stimulation of PFK-1 and inhibition of Fru-1,6-BPase reducing futile cycling (increased glucose entry or, in muscle, glycogen breakdown)
• Glycolysis inhibited by decreased Fru-2,6BP by stimulation of Fru-1,6-BPase and inhibition of PFK-1 (reduced glucose entry or, in muscle, decreased glycogen breakdown)

Question 16

Explain how F-2,6-BP is a problem in the liver ?

Answer 16

Liver: uses glucose produced via gluconeogenesis and glycogen to maintain blood glucose and glycolysis is inhibited – need to avoid F6P produced by gluconeogenesis leading to increased F-2,6-BP levels

Question 17

Explain the the bifunctional enzyme in the liver ?

Answer 17

In liver, not only have to control glycolysis at the level of PFK-1, but also the reverse reaction of gluconeogenesis at F-1,6,BPase to allow reciprocal control of the two reactions

Question 18

What kind of enzymes are PFK-2 and F-2,6-BPase in the liver ? and how are they controlled ?

Answer 18

They are a single tandem enzyme with two active sites:
• N-terminal kinase domain
• C-terminal phosphatase domain

• These are controlled hormonally via phosphorylation

Question 19

What does the Hormonal regulation of glycolysis and gluconeogenesis depend on?

Answer 19

The phosphorylation state of

PFK-2/FBPase-2

Question 20

What does Insulin signalling activate ?

Answer 20

It activates the PFK-2 domain, increasing glycolysis

Question 21

What does Glucagon signalling activate ?

Answer 21

The FBPase-2 domain, increasing gluconeogenesis

Question 22

High glucose which leads to high F6P, stimulates and inhibits ? (liver)

Answer 22

• Stimulates PFK2

- Inhibits F-2,6-BPase

Question 23

Citrate spilling out of the mitochondria during acetylCoA overload (e.g. starvation) will ?

Answer 23

Inhibit PFK2 (and PFK1): promote gluconeogenesis

Question 24

What is PEP and what does it inhibit ?

Answer 24

• PEP, one of the first products of gluconeogenesis

- Inhibits PFK2

Question 25

Influx of glucose into the liver after high carbohydrate meal stimulates ?

Answer 25

Both glycolysis and the

pentose phosphate pathway (PPP)

Question 26

What does PPP produce ? and what does it do ?

Answer 26

• PPP produces xylulose-5-phosphate, which is an activator of PPase-2A
• Dephosphorylates PFK-2/F-2,6-Bpase increasing Fru-2,6-BP

Question 27

What are the 3 main control points of glycolysis ?

Answer 27

1. The hexokinase/glucokinase step
   - In the muscle, high affinity hexokinase not regulated: glucose will be phosphorylated to G6P and used in glycolysis or converted to glycogen for storage
   - In the liver, low affinity glucokinase will only be activated at high levels of blood glucose after a meal
2. The key control point at the committed step: PFK1
3. The pyruvate kinase step, converting PEP to pyruvate

Question 28

Explain the last reaction of glycolysis of the different forms in liver (L-type) and muscle (M-type)

Answer 28

• M-type is a Michaelis-Menten enzyme controlled only by [PEP] and [ADP]
• L-type is an allosteric enzyme, also subject to reversible phosphorylation. Needs to be switched off during gluconeogenesis to prevent futile cycling and conversion of newly formed PEP back to
  pyruvate. Switched on during increased glucose entry for lipid synthesis

Question 29

What is the allosteric control of Pyruvate Kinase ?

Answer 29

• Stimulated by: fructose-1,6-bisphosphate (feedforward from PFK-1 during periods of increased glucose entry – fat synthesis)
• Inhibited by: alanine and ATP (major gluconeogenic substrate)

Question 30

Explain the regulation of L-pyruvate kinase in terms of inhibition and activation ?

Answer 30

• Short term inhibition: Phosphorylation via glucagon & adrenaline or increased supply of gluconeogenic substrates in the form of alanine and ATP
• Short term activation by: dephosphorylation via insulin or increased glucose supply by F-1,6-BP (overcomes effect of phosphorylation, ATP and alanine)