Lecture 4: Glycolysis and TCA Cycle Flashcards
Give the reactions that make up glycolysis, with the enzymes which catalyse them.
1) Glucose is phosphorylated to Glucose-6-phosphate by hexokinase, with the simultaneous hydrolysis of ATP.
2) Glucose-6-phosphate is isomerised to Fructose-6-phosphate by phosphoglucoisomerase.
3) Then Fructose-6-phosphate is phosphorylated to Fructose-1,6-bisphosphate by Phosphofructokinase, with the simultaneous hydrolysis of ATP.
4) Fructose-1,6-bisphosphate is then cleaved to two triose phosphate called Glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, by aldolase.
5) Dihydroxyacetone phosphate is then isomerised to Glyceraldehyde-3-phosphate by triose phosphate isomerase, so that there are now 2 Glyceraldehyde-3-phosphate molecules.
This is the end of the energy investment phase. Now the energy generating phase begins.
6) 2 Glyceraldehyde-3-phosphate are converted to 2 1,3-bisphosphoglycerate, which is the first high energy compound in glycolysis (it is an acyl phosphate). The reaction is catalysed by Glyceraldehyde-3-phosphate dehydrogenase and requires NAD+ + Pi —> NADH + H+.
7) 2 1,3-bisphosphoglycerate are converted to 2 3-Phosphoglycerate in the first substrate level phosphorylation reaction (ADP + H+ –> ATP). This reaction is catalysed by Phosphoglycerate kinase.
8) 2 3-Phosphoglycerate are isomerised to 2 2-Phosphoglycerate by Phosphoglucomutase.
9) 2 2-Phosphoglycerate is dehydrated to 2 Phosphoenolpyruvate, another high energy compound. Water is lost and the reaction is catalysed by Enolase.
10) 2 Phosphoenolpyruvate are converted to 2 Pyruvate in the second substrate-level phosphorylation reaction (ADP + H+ —> ATP). The reaction is catalysed by Pyruvate kinase.
Which of the reaction in glycolysis are highly favourable? What is the two other feature these reactions have in common?
Reactions 1, 3 and 10.
They are all essentially irreversible.
They are regulation points and their enzymes are allosterically regulated (hexokinase, phosphofructokinase and pyruvate kinase).
What allosteric regulation of glycolysis is there?
The main targets of allosteric regulation are the enzymes catalysing irreversible reactions: hexokinase, phosphofructokinase and pyruvate kinase.
- Hexokinase is allosterically inhibited by Glucose-6-phosphate, the product of the reaction it catalyses. If there is a build up of Glc-6-P, glycolysis is slow and hexokinase does not need to work hard.
- Phosphofructokinase catalyses the committed step of glycolysis so is the most important point of regulation. It is inhibited by ATP, Citrate and Lactate. It is activated by AMP and Fructose-2,6-bisphosphate (a regulatory molecule made by an enzyme (Fructose-2,6-bisphosphatase) which is activated by glucagon and inhibited by insulin).
- Pyruvate kinase is activated by fructose-1,6-bisphosphate and inhibited by ATP and alanine.
What hormonal control of glycolysis is there?
Fructose-2,6-bisphosphate, which activates phosphofructokinase, is regulated by phosphorylation. It is phosphorylated/dephosphorylated by Fructose-2,6-bisphosphatase, which is regulated by hormones (glucagon/adrenaline and insulin).
Glucagon causes the enzyme Fructose-2,6-bisphosphatase to be phosphorylated, which makes it converted Fructose-2,6-bisphosphate to fructose-6-phosphate. This means glycolysis is not activated and slows, so less glucose is used up. (Insulin causes the reverse)
Pyruvate kinase is also hormonally regulated. It is phosphorylated by glucagon, which inactivates it. Glycolysis then slows and less glucose is used up.
Give a non-balanced reaction to show what is used and what is made in glycolysis.
Glucose + 2 ADP + 2 Pi + 2 NAD+ —> 2 Pyruvate + 2 ATP + 2 NADH
Where does glycolysis take place?
In the cytoplasm
Discuss the permeabilities of the mitochondrial membranes.
The outer mitochondrial membrane is permeable to molecules up to 10 kDa in size. The inner mitochondrial membrane is impermeable.
Where does the TCA Cycle take place?
In the mitochondrial matrix
What conditions are necessary for the TCA cycle to take place?
Aerobic conditions (oxygen must be available)
What happens after pyruvate enters the matrix?
After pyruvate enters the mitochondria, it is converted to acetyl CoA by an enzyme system called pyruvate dehydrogenase complex.
Pyruvate + NAD+ + CoASH —> Acetyl CoA + NADH + CO2
Describe the steps of the TCA Cycle.
1) Oxaloacetate and acetyl CoA combine to make citrate. CoASH is regenerated and leaves and the enzyme is citrate synthase.
2) Citrate is dehydrated to cis-aconitate, which is hydrolysed again to form isocitrate. The enzyme is called aconitase (contains an iron-sulfur centre) and the overall reaction is an isomerisation.
3) Isocitrate then undergoes oxidative decarboxylation to a-ketoglutarate, catalysed by isocitrate dehydrogenase. NAD+ —> NADH + H+ and CO2 is released.
4) a-ketoglutarate is then converted to succinyl CoA by a-ketoglutarate dehydrogenase. NAD+ —> NADH + H+. CO2 is released, CoA is used. Succinyl CoA has high energy bond.
5) Succinyl CoA is converted to succinate in a substrate-level phosphorylation. GDP + Pi —> GTP + CoASH. Then ADP + GTP —> ATP + GDP (catalysed by nucleoside diphosphate kinase).
6) Succinate is converted to fumurate, catalysed by succinate dehydrogenase. FAD —> FADH2
7) Fumurate is converted to malate by an enzyme called fumurase. This reaction involves the addition of water.
8) Malate is then converted to oxaloacetate, by malate dehydrogenase. NAD+ —> NADH + H+.
What is produced every turn of the TCA cycle, in terms of coenzymes?
2 CO2, 3 NADH, FADH2, GTP
How many electrons does NAD+ accept and in how many steps?
2 electrons in one step
How many electrons does FAD accept and in how many steps?
2 electrons in two, one electron, steps
What is a semiquinone?
A free radical intermediate when only one of two electrons has been accepted. Upon accepting a second electron, the semiquinone becomes a quinone/quinol. Examples: Coenzyme Q and FAD
What is the difference between NAD+ and NADP+?
NAD and NADP have the same molecular structure, but NADP has a phosphate group on its adenine. NAD is important in respiration and NADP is important in reductive biosynthesis, such as fatty acid synthesis, and in plant metabolism.
What is NAD synthesised from?
A vitamin called Niacin in the form of nitratinic acid/nicotinamide.
What is FAD synthesised from?
Vitamin B2, riboflavin
