Citric acid cycle and Oxidative Phosphorylation

Question 1

GLUCOSE TO PYRUVATE METABOLISM

Answer 1

Glucose to pyruvate metabolism, an anaerobic process, yields a fraction of the ATP available from glucose (2 ATP).
Aerobic processing produces more ATP.
Process begins with the complete oxidation of glucose derivatives to carbon dioxide
This oxidation takes place via the citric acid cycle, also known as the tricarboxylic acid (TCA) cycle or the Krebs Cycle.
The citric acid cycle is the final common pathway for the oxidation of fuel molecules (carbohydrates, fatty acids and amino acids).Most enter the cycle as acetyl coenzyme A (Acetyl CoA).

Question 2

Net result of the citric acid cycle

Answer 2

One turn of the cycle produces:
4 molecules of reduced cofactor (3 NADH2, and 1 FADH2).
1 molecule “high energy” phosphate (1 guanosine Triphosphate (GTP), an ATP equivalent).
2 carbon atoms (as CO2). (NOTE: input was x2 C from the acetyl).

Question 3

Energy powerhouse

Answer 3

Acetyl CoA is generated from the oxidative decarboxylation of pyruvate.
Under aerobic conditions, the reactions of this process, citric acid cycle, take place in the mitochondria of eukaryotes. In contrast with those of glycolysis that take place in the cytoplasm

Question 4

STEPS OF THE KREBS CYCLE (citric acid cycle)

step 1:

Answer 4

This reaction is catalyzed by citrate synthase.
Oxaloacetate + Acetyl CoA➡️ Citryl CoA➡️Citrate

Citryl CoA, the intermediate, is a key energy-rich molecule (thioester). The hydrolysis of Citryl CoA to citrate and CoA drives the overall reaction in the direction of citrate.

Question 5

Step 2

Answer 5

This reaction is catalyzed by aconitase.

Question 6

Step 3

Answer 6

The oxidative decarboxylation of isocitrate is catalyzed by Isocitrate dehydrogenase. The first of four oxidation-reduction reactions.

This oxidation generates the first high transfer potential electron carrier, NADH, in the cycle.

Question 7

step 4

Answer 7

A further oxidative decarboxylation generates succinyl CoA from ALPHA-ketoglutarate, catalyzed by ALPHA-ketoglutarate dehydrogenase complex.

ALPHA-Ketoglutarate + NAD+ +CoA ➡️ Succinyl CoA+ NADH + CO2

Succinyl CoA is an energy rich thioester compound with a comparable hydrolysis to ATP (-31 kJ mol-1) of -33.5 kJ mol-1.

Question 8

Step 5

Answer 8

This reaction is catalyzed by ALPHA-succinyl CoA synthase.

The cleavage of the thioester in succinyl CoA is coupled to the phosphorylation of a purine nucleoside diphosphate (usually GDP).

Question 9

Step 6 and 7

Answer 9

The final stages of the cycle are reactions of 4C compounds and constitute the final stages of the citric acid cycle: regeneration of oxaloacetate.

Succinate is oxidised to fumarate by succinate dehydrogenase. Malate is oxidised to form oxaloacetate, catalysed by malate dehydrogenase

Question 10

Stiochiometery of the Citric Acid Cycle

Answer 10

The balance equation for the complete oxidation of pyruvate (last stage of glycolysis) to CO2 and H2O in the citric acid cycle is:
CH3—CO—COOH + 2.5O2➡️3CO2 + 2H2O

Question 11

Carbon dioxide

Oxygen

Answer 11

There are three decarboxylations reactions (pyruvate, isocitrate,ALPHA-ketoglutarate). Therefore CO2 balances.

2.5 molecules of O2 represent five atoms of oxygen. There a five oxidation steps:
Oxidative decarboxylation of pyruvate (NAD+).
Oxidation of isocitrate (NAD+).
Oxidation of ALPHA-ketoglutarte (NAD+).
Oxidation of succinate (FAD).
Oxidation of malate (NAD+).
Oxidation of the reduced products of these cofactors (NADH and FADH) each require an atom of oxygen = 5 in total. Therefore the equation balances.

Question 12

Water

Answer 12

The equation predicts that 2 molecules of water are produced.
However, the 5 atoms of oxygen will generate five molecules of water when oxidising the 5 reduced co-factors. To balance this up, we should find 3 reactions in which a molecule of water is consumed, They are:
Condensation of acetyl CoA with oxaloacetate.
Hydration of fumarate to malate.
Cleavage of succinyl phosphate.

Question 13

Citric acid effectiveness

Production of energy from 1 glucose

Answer 13

Production of energy from 1 glucose
Only one molecule of high-energy phosphate (GTP) is produced per turn of the cycle (i.e. two per molecule of acetyl Co-A fed in from glycolysis).
But there are 3 NADH and 1 FADH2.
The generation of ATP by oxidation of these cofactors is called oxidative phosphorylation. This yields a great deal of energy: ~2.5 ATP produced per molecule of NADH, ~1.5 per FADH2 and 2 per GTP.

This amounts to (5* NADH x 12.5 ATP) + (1 FADH x 1.5 ATP) ATP per turn = 14, or 28 per molecule of glucose. Together with the 4 from GTP, this amounts to ~32 per molecules of glucose.
Including 2 NADH from glycolysis
Citric Acid cycle of ATP per molecule of glucose is ~32
Adding in the 2 ATP (net) produced in glycolysis, This adds up to 34 molecules.
GRAND TOTAL of ATP per molecule of glucose is ~34

GRAND TOTAL of ATP per molecule of glucose is 34.
An ATP is worth 31 kJ mol-1.
Total energy capture = 34 x 31 = 1054 kJ per mol glucose.
A mol of glucose has –2880kJ/mol. % efficiency is 1054/2880 x 100 = 37%

Question 14

citric acid effectiveness

Generation of reduced cofactors

Answer 14

The cycle yields 3 NADH and 1 FADH2 per turn of the cycle, i.e. 8 per molecule of glucose. This is potentially available for biosynthetic purposes.

HOWEVER: if the reduced cofactors are used in the production of ATP by oxidative phosphorylation, then they are not available as reducing power for biosynthesis.

Question 15

citric acid effectiveness

Provision of metabolic intermediates

Answer 15

There are several intermediates in the citric acid cycle that are potentially useful to “tap-off” as precursors for other processes. For example, ALPHA-ketoglutarate and oxaloacetate are easily transformed into amino acids and can enter protein metabolic processes. Succinyl-CoA is the starting point in the biosynthesis of porphyrins.

Question 16

citric acid effectiveness

Provision of metabolic intermediates
HOWEVER:

Answer 16

HOWEVER: remember that the net input to the cycle is 2 carbon atoms (from acetyl CoA). Both of these are decarboxylated and lost as CO2 in the breath.
The 2 carbon atoms of Acetyl-CoA are therefore “doomed” to decarboxylation. Removal of a molecule of one of the intermediates will therefore grind the cycle to a halt.
This can be remedied by “topping-up”, i.e. inserting more carbon from the glycolysis pathway from a position before the decarboxylation of pyruvate
These “topping-up reactions” are called anaplerotic reaction

Question 17

anaplerotic reactions

Answer 17

There are several of these anaplerotic reactions. Here is one in which pyruvate is carboxylated to oxaloacetate:
CO2 + CH3CO.COOH + ATP + H2O➡️(Pyruvate carboxylase)➡️ COOH.CO.CH2COOH + ADP + Pi
The oxaloacetate can now enter the citric acid cycle again.

Question 18

OXIDATIVE PHOSPHORYLATIONOut and in of the matrix

Answer 18

A flow of electrons from NADH and FADH2 to O2.
This takes place through protein complexes in the mitochondrial inner membrane and leads to the pumping of electrons out of the mitochondrial matrix.

The resulting unequal distribution of protons generates a pH gradient and a tans-membrane electrical potential that creates a proton-motive force.

ATP synthesis is generated when protons flow back into the matrix through an enzyme complex.

Question 19

Ion Gradients Across Membranes (IGM)

Answer 19

The electrochemical potential of IGAM’s (produced by the oxidation of fuel molecules) ultimately powers the synthesis of most ATP in the cells.

Versatile:
couple thermodynamically unfavourable reactions to favourable ones.
Productive:
- 90% of ATP generation (oxidative phosphorylation)
- proton gradients can drive ATP syntheis when protons flow through an ATP synthesising enzyme (i.e. in mitochondrial membranes).

Question 20

Phosphoryl-transfer

Answer 20

The final stage of oxidative phosphorylation is carried out by ATP synthase.
This is a an ATP synthesising assembly that is driven by the flow of electrons back into the mitochondrial matrix.

Four pairs (8 e-) of electrons are transferred: three to NAD+ and one to FAD) for each acetyl groups that is oxidised. Then, a proton gradient is generated as electrons flow from the reduced forms of these carriers to O2: THIS GRADEIENT IS USED TO SYNTHESISE ATP