The TCA cycle

Dr. S. PicksleyH17, ext 5935

Petrol or diesel(hydrocarbons)

+ oxygen (O2)

+ spark

Energy + C02 + H20

Combustion releases energy very fast & explosively.We need to release energy from food slowly and discretely,This is done by the TCA cycle and oxidative phosphorylation.

THE PROBLEM

Learning objectives

1. Understand how complex foodstuffs (carbohydrate, fat & protein) are converted into energy (ATP).

2. The conversion of food to ATP is a two step process, in which catabolites (breakdown products of carbohydrate, fat & protein) are channelled into the TCA cycle and oxidised to produce C02 and energy rich (reduced) molecules of NADH and FADH2 (nucleoside cofactors) and GTP.

Learning objectives cont.

3. NADH and FADH2 then transfer their energy to ATP by electron transport / oxidative phosphorylation (the subject of the next lecture!).

4. In effect these two processes represent RESPIRATION - the process by which aerobic cells obtain energy from the oxidation of food by oxygen.

5. The TCA cycle is also a starting point for some biosynthetic pathways (anabolic metabolism, - from simple to complex molecules).

Recommended texts

‘Instant notes in biochemistry’ Section L1The Citric acid cycle. By Hames et al. p291-305.

‘Biochemistry’ by Campbell. p532-571.

OverviewWe derive all our cellular energy from carbohydrates, fat and proteins.  Carbohydrates, fat and proteins, are catabolised (broken down) to a common intermediate, acetyl CoA, that enters the TCA cycle. CoA stands for a co-enzyme A (an essential enzyme activator).

By a series of enzyme catalysed reactions the two carbon atoms of acetyl CoA is oxidised (loses electrons) to C02 to produce GTP (from GDP) and four pairs of electrons.

These electrons are transferred to nucleotide derived coenzymes, NAD+ and FAD, which finally transfer them to O2 (oxygen).

Overview cont.Energy is produced and trapped as ATP by oxidative phosphorylation. Energy is also produced during the TCA cycle in the form of GTP (which is formally equivalent to ATP). Energy use in man At rest we will consume half our body weight in ATP per day! Of course we cannot store this amount of ATP. As we consume energy, ATP --> ADP + Pi, we replace it by oxidising food molecules, and regenerating ATP molecules.  The production of ATP is initially carried out by enzymes of the TCA pathway and finally by an electron transport chain.

The TCA or Citric acid or Krebs Cycle

Its called a cycle because the acetyl CoA reacts with a metabolite (oxaloacetate), that is regenerated by a series of enzyme catalysed reactions.

Oxaloacetate + acetyl Coa ------> citrate

Hence the alternative name, the Citric Acid Cycle.

Citrate has three COO- groups, TriCarboxylic Acid.

The cycle takes place in the mitochodria in all mammalian cells.

TCA cycleLocation: occurs in the mitochondria of eukaryotic cells and in the cytosol of bacteria.

Glycolysis Glucose ---> Pyruvate-------> Acetyl CoA  Fats, Proteins ------> Acetyl CoA  Acetyl CoA is the form in which fuel molecules enter the cycle.  This cycle has eight well-characterised stages.

We will only consider an overview of the cycle.

TCA cycle Acetyl CoA (C2) + oxaloacetate (C4) --> Citric acid (C6)

Oxaloacetate (C4) + 2CO2

And then the cycle begins again. 2 carbon atoms enter and 2 leave.  Yields per cycle:

1 GTP, 3NADH, 1FADH2, CoA + 2CO2

In the text books it will describe the TCA cycle as follows:AcetylCoA

Citrate

Isocitrate

A-ketoglutarate

Succinyl CoA

Succinate

Fumarate

Malate oxaloacetate

THIS LEVEL OF DETAIL IS NOT REQUIRED. THE AIM IS TO UNDERSTAND THE PRINCIPLES.

TCA cycle key points

Acetyl CoA + oxaloacetate -----> Citric acid 2 Carbons + 4 Carbons 6 Carbons

2 CO2

Yields: 1 GTP, 3 NADH, 1 FADH2, CoA + 2CO2

Acetyl CoA is oxidised to CO2

This does not involve oxygen

Oxidation of acetyl CoAAcetyl CoA is oxidised to CO2 by the donation of electrons (e-) along with the hydrogen (H+) or hydride (H-)ion.

H atom = H+ + e- (electron)

Hydrogen ion or proton is H+ Hydride ion is H-, H- = H+ + 2e-

Oxidation = loss of electrons. Reduction = gain of electrons.

Oxidation and reduction occur side by side, as electrons are not created or destroyed

Electrons are donated to NAD+ or FAD

NAD is nicotinamide adenine di-nucleotide.

It is a co-enzyme, i.e. a chemical that is essential for enzyme activity but is easily dissociated from the protein with the loss of activity.

NAD+ + 2H+ + 2e- = NADH + H+ (oxidised form) (reduced form)

FAD is flavin adenine di-nucleotide. It is a prosthetic group, i.e. a chemical that is essential for activity and that is physically linked to an enzyme.

FAD + 2 H+ + 2e- = FADH2 (oxidised form) (reduced form)

Oxidised and reduced forms of NAD+ and FAD

The TCA reactionAcetyl CoA + 3NAD+ + 1FAD + GDP + Pi + 2H20

CoA + 2CO2 + 3NADH + 3H+ + 1FADH2 + GTP.  One complete cycle yields in energy terms: 1 GTP + 3NADH + 1FADH2. (=ATP) GTP is a nucleotide and an energy source just like ATP and is equivalent to ATP.

The production of GTP during the TCA cycle is referred to as substrate level phosphorylation, i.e. it is directly produced from GDP + Pi, without an intermediate.

NADH & FADH2 are energy-rich carrier molecules

NADH and FADH2 are energy-rich molecules, which transfer their energy to ATP molecules by an indirect route of transfering electrons via the electron transport chain to oxygen (oxidative phosphorylation).  1 molecule of NADH ------> 2.5 molecules of ATP

(3 molecules of ATP in older textbooks) 1 molecule of FADH2 ------> 1.5 molecules of ATP (2 molecules of ATP in older textbooks)

Regulation of the TCA cycle by respiratory control

The flow of intermediates through the TCA cycle isregulated by the demand for ATP.

If energy demand is high as indicated by low

[ATP]:[ADP] and [NADH]:[NAD+]

there will be a high flow of intermediates through the TCA cycle to produce the required energy.

TCA cycle as a source of biosynthetic precursors

The TCA cycle in addition to generating energy richmolecules also has a role in generating important precursors for: 1. Synthesis of some amino acids2. Synthesis of glucose3. Synthesis of proteins4. Synthesis of nucleic acids5. Syntheis of fats 6. Synthesis of pophyrins (used to make haem for

haemoglobin).