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CELLULAR RESPIRATION. Chapter 19 & 20. Biochemistry by Campbell and Farell (7 th Edition) By Prof M A Mogale

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(1)

CELLULAR RESPIRATION

Chapter 19 & 20

Biochemistry by Campbell and Farell

(7

th

Edition)

(2)

1. Cellular respiration (energy capture)

 The enzymatic breakdown of food stuffs in the presence of oxygen to produce cellular energy (ATP)

(3)

STAGE 1

 Conversion of energy-rich food stuffs into acetyl CoA.

 Different for carbohydrates (glycolysis), proteins and lipids (β-oxidation)

 Very little or no ATP is formed during this stage  Energy released is stored as NADH and FADH2

(4)

STAGE 2 (Citric acid cycle)

• Aerobic oxidation of acetyl CoA to CO2. in mitochondria (8 steps) • Common for carbohydrates, amino acids and fatty acids

• Acetyl CoA (2C) combine with oxaloacetate (4C) to form citric acid (6C) • Citric acid is gradually oxidized to regenerate oxaloacetate

• Energy released is stored as NADH and FADH2,

(5)

 The individual reactions of the citric acid cycle

 Phase 1 : Introduction and loss of two carbon atoms (Steps 1–4) Step 1: Condensation of OA and Acetyl CoA to form citrate and CoA

 The reaction is catalysed by the enzyme citrate synthase (condensing enzyme)

 A synthase is an enzyme that make a new covalent bond during the

reaction but does not require the direct input of ATP

 The reaction is an exergonic reaction ΔGo = -32.8 kJ/mol (energy

(6)

Step 2: Isomerization of Citrate to isocitrate

 Catalysed by the enzyme aconitase that utilize an Fe2+-- sulphur

cluster as a cofactor

 In this reaction a symmetrical (achiral) compound is converted to a

chiral compound (a secondary alcohol) yielding several possible isomers

 The reaction proceed via an enzyme

(7)

 Aconitase is the target site for the toxic action of fluoroacetate, a plant product that has been used as a pesticide

 Fluoroacetate is a suicide enzyme (trojan horse) inhibitor which is converted in to the toxic inhibitor fluorocitrate in the active side of the enzyme by the enzymes acetyl CoA synthetase and citrate synthase

 The action of fluoroacetate is similar to that of the legendary trojan horse

(8)

Step 3: Oxidative decarboxylation of isocitrate to alpha-ketoglutarate (First oxidation)

• The reaction is the first of the

two oxidative decarboxylation of the citric acid cycle

• The reaction which proceed in

two steps is catalysed by the enzyme isocitrate

dehydrogenase

• One molecule of NADH and one

molecule of CO2 are produced during this stage

• One mole of NADH will

eventually produce approxi-mately 2,5 mole of ATP when it donates its electron to O2

during oxidative phosphorylation

(9)

Step 4: Formation of Succinyl-CoA and CO2 from alpha-ketoglutarate (Second oxidation step)

• The reaction proceed in several steps and is catalysed by a

multienzyme system known as alpha-ketoglutarate dehydrogenase complex

• The alpha-ketoglutarate dehydrogenase enzyme complex utilizes thiamine pyrophosphate(TPP), FAD, lipoic acid and Mg2+ as enzyme

(10)

Step 4: Formation of Succinyl-CoA and CO2 from alpha-ketoglutarate (Second oxidation step)

(11)

• The reaction is similar to the one catalysed my pyruvate dehydrogenase complex that convert pyruvate into acetyl CoA.

• It was initially believed that the two carbon atoms lost as CO2 in step 3 and 4 of the CA cycle were the acetyl CoA carbons, however current experimental evidence (isotope tracing) show that this carbon atoms come from oxaloacetate

• The conversion of alpha-ketoglutarate to succinyl CoA is highly exergonic ( Go = -33.4 kJ/mol = -8.0 kcal/mol)

(12)

 Phase 2: Regeneration of oxaloacetate

 Two carbons have entered the CA cycle as acetyl CoA, and at this stage two have been lost as CO2

 In the remaining reactions, the four carbon intermediate ,

succinyl-CoA is converted to oxaloacetate in four steps (steps 5 -8), two of them involving dehydrogenation reactions

(13)

Step 5: Formation of Succinate (Substrate level phosphorylation)

 The reaction is catalysed by the enzyme succinyl-CoA synthetase

 A synthetase is an enzyme that creates a new covalent bond and requires a direct input of energy from a compound with a high phosphate transfer potential

 The free energy of hydrolysis of succinly-CoA to produce succinate is -33.4 kJ/mol

(14)

 Thus this reaction, cannot be empowered by hydrolysis of ATP to produce ADP + Pi

 The energy required for this reaction is provided by the

hydrolysis of the thioester bond of succiinyl-CoA to produce succinate and CoA-SH

 In this reaction the energy resulting from thiolysis (forward reaction) is also used to form GTP from GDP

 Note that the name of the enzyme describe the reverse

reaction in which GTP is hydrolysed to produce GDP thereby releasing energy to form the thoester bond

 This reaction step is referred to as substrate level

phosphorylation to distinguish it from formation of ATP coupled to oxidative phosphorylation

(15)

 In mammals, the GTP produced in the succinate synthetase reaction can exchange its terminal phosphoryl group with ADP to yield ATP via a reaction catalysed by the enzyme

(16)

Step 6: Flavin-Dependent Dehydrogenation

 Completion of the cycle involves conversion of the four-carbon

succinate to the four-carbon oxaloacetate

 The first of the remaining reactions is the FAD-dependent

dehydrogenation of two saturated carbon atoms to produced a

double bond catalysed by succinate dehydrogenase

 Succinate dehydrogenase is an inner mitochondrial

membrane-bound enzyme that is part of the electron transport chain involved in oxidative phosphorylation

(17)

 The reaction (the oxidation of an alkane to an alkene) is not sufficiently exergonic to reduce NAD, but it does yield

enough energy to reduce FAD

 In general, FAD is a better oxidising agent than NAD+ and

NADH is a better reducing agent than FADH2

 The action of succinate dehydrogenase is stereo selective, forming only the trans isomer, fumarate

(18)

Step 7: Hydration of the carbon-carbon double bond

 Fumarate is converted to L-malate by stereospecific addition of components of a water molecule across the double bond by the enzyme fumarate hydratase (fumarase)

 This reaction is highly exergonic in the foward direction and has an equilibrium constant of about 4

(19)

Step 8: Conversion of malate to oxaloacetate

 This is the final step of the citric acid cycle catalysed by the malate dehydrogenase enzyme

 The oxidation of alcohols to ketone or aldehyde groups are more energetically favourable and provide sufficient energy

(20)

 Stoichiometry of the citric acid cycle

• The cycle started when a two carbon acetyl-CoA combined

with a four carbon oxaloacetate to produce citrate

• Two carbon atoms were removed as carbon dioxide as citrate was further metabolized

• Four oxidation reactions occurred during the cycle, with NAD+

serving as an electron acceptor in three and FAD for the fourth • One high energy phosphate was generated by the reaction

catalysed by succinyl CoA synthetase

Acetyl-CoA + 3NAD

+

+ FAD + GDP + P

i

+ 2H

2

O

2CO

2

+ 3NADH +3H

+

+ FADH

2

+ + GTP + CoASH

(21)

 Energetics of the citric acid cycle

• Although some individual steps in the citric acid cycle may be endergonic the overall reaction of the cycle is exergonic (Table 19.2, C & F, Page 547) ΔGo = - 40 kJ/mol

• In terms of ATP production a total of 9 ATP molecules are produced per one turn of cycle

Pathway Substrate-Level Phosphorylation Oxidative Phosphorylation Total ATP

Krebs Cycle 1 ATP 3 NADH = 6 ATP

1 FADH2 = 2 ATP 9

(22)

 Regulation of the citric acid cycle

• Regulation of the citric acid cycle occurs both at the level of entry

of fuel in to the cycle and by control of key reactions within the cycle

• The most important factor controlling the citric acid cycle is the

relative intra-mitochondrial concentration of NAD+ and NADH

• Key sites for allosteric regulation are the reactions catalysed by

(23)
(24)

 Anaplerosis and Cateplerosis

• Most citric acid intermediates are used as biosynthetic intermediates and hence may be depleted

• Anaplerosis is a process whereby citric acid intermediates

used in biosynthe tic pathways are replenished

• Anaplerotic pathways may be classified into three groups, those replenishing oxaloacetate, those replenishing malate and those involving transamination of amino acids

• Cateplerosis is the oposite of anaplerosis i.e. pathways that

drain citric acid intermediates from the cycle for use in biosynthetic pathways

(25)
(26)

 Anaplerotic pathways that replenish oxaloacetate

• In mammals, the most important anaplerotic pathway for

generating oxaloacetate is the reversible ATP-dependent

carboxylation of pyruvate to give oxaloacetate

• This reaction is catalysed by the enzyme pyruvate

(27)

 Anaplerosis of malate

• The anaplerotic pathway for malate involves the malic

enzyme (malate dehydrogenase)

• This enzyme catalyses the reductive carboxylation of

(28)
(29)
(30)
(31)

 Role of citric acid cycle in lipogenesis and pyruvate and

(32)

 The Glyoxylate Cycle: An Anabolic Varient of the Citric

Acid Cycle

 One of the fundamental differences between plant and animal cells is that plants (and some microorganisms) can synthesize carbohydrates (glucose) from fat

 The conversion of fat into sugars is crucial in the development (germination) of seeds

 When seed germinate, triacylglycerols are broken down and converted into sugars, a process which provide the energy and raw materials for the growth of the plants

 Plants synthesize sugars from fats by means of the glyoxylate

cycle which is considered as an anabolic variant of the citric acid cycle

(33)

The Glyoxilate Cycle (Cont….)

• The glyoxylate cycle occurs in the glyoxysome, a specialized plant organelle that caries out both beta-cell oxidation of fatty acids to acetyl CoA and utilization of acetyl CoA in the

glyoxylate cycle

• In the glyoxylate cycle, acetyl CoA (provided by the beta- oxidation of fatty acids or by acetate thiokinase) reacts with oxaloacetate to give citrate, which is converted to isocitrate by the enzyme aconitase

• At this point, the glyoxylate cycle diverges from the citric acid cycle

• The next reaction is catalysed by isocitrate lyase, which

cleaves isocitrate to glyoxylate and succinate

• Glyoxylate then accepts acetate form another cellular acetyl-CoA to produce malate in a reaction catalysed by malate

(34)
(35)

 The Link between the Citric Acid cycle and molecular

Oxygen

• The citric acid cycle does not operate under anaerobic conditions

• This is because of the fact that the citric acid cycle is regulated among other things, by the concentration of NADH produced by the cycle

• After being produced by the citric acid cycle, NADH (and FADH2) donates its electrons to molecular oxygen through the respiratory chain

• Thus, in the absence of oxygen NADH will accumulate and inhibit the citric acid cycle

References

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