Krebs cycle or Citric acid cycle

· Named after German-British scientist and Nobel prize winner Hans Krebs, who unraveled the cyclical nature of this central part of cellular respiration.

- the unraveling of the intricate details and cyclical nature of the Krebs cycle is hailed as one of the greatest achievements in the biological sciences

- It is a cyclical, 8-staged chemical process which is started by entering of Acetyl-CoA into the cycle.

- The 8 stages of the Krebs or Citric Acid cycle

1. REACTIONa. Co-enzyme A (HS-CoA) is cleaved from acetyl-CoA and recycled; b. only the 2-carbon molecule acetic acid (C2) enters the cycle; c. the C2 compound is covalently combined with the 4-carbon molecule oxal acetate (OxAc) to form citric

acid (= C6 compound!), which gets the energy of the acetyl group.

- since citric acid (or citrate) is the first chemical product, the Krebs cycle is also often referred to as the citric acid cycle- this rate-limiting chemical reaction is a typical condensation reaction which is catalyzed by the enzyme citrate synthase- citrate synthase is a critical regulatory enzyme of the Krebs cycle

2. REACTIONThe enzyme aconitase catalyzes the reversible transformation of citrate into isocitrate by reversible addition of water.- this chemical reaction is an addition reaction- aconitase is an iron-containing enzyme with a iron-sulfur (Fe-S) cluster

- know we know why iron is an important trace element crucial for energy conversion processes in cells!

3. REACTIONIn the next step isocitrate is dehydrogenated (loss of two hydrogens) and simultaneously decarboxylated (loss of CO2) to the 5-carbon molecule alpha-ketoglutamic acid (= a-KG or a-ketoglutarate).- this oxidative decarboxylation reaction is catalyzed by the enzyme isocitrate dehydrogenase, an enzyme which contains NAD+ as co-enzyme and requires magnesium (Mg2+) or manganese (Mn2+) as co-factors

4.REACTIONIn step number 4, α-ketoglutarate is oxidized to succinyl-CoA and CO2 under formation of the redox molecule NADH + H+ by the catalytic action of the a-ketoglutarate dehydrogenase enzyme complex;

- along the reaction, a high-energy bond ( symbolized by “~” in the Graphic below) between one carboxyl group of ketoglutarate and coenzyme A is formed.- the chemical reaction is an oxidative decarboxylation reaction which is similar to the one catalyzed by the pyruvate dehydrogenase complex (see above)- the enzyme complex is comprised of three sub-units and requires thiamine pyrophosphate (TPP), Mg2+, coenzyme A, FAD, and lipoic acid

5. In step, the highly energized succinyl-CoA molecule is converted into succinate in a coupled hydrolysis reaction which is catalyzed by the enzyme succinyl-CoA synthetase- succinyl-CoA synthetase couples the highly exergonic hydrolysis of coenzyme A from succinyl-CoA with the endergonic formation of GTP from the recursor molecule GDP;- cells are able to convert GTP into ATP in the presence of ADP with the help of an enzyme called nucleoside diphosphokinase (NDK);

(NDK)

GTP + ADP → GDP + ATP

- this is the only step of the Krebs cycle were substrate level phosphorylation takes place and a net production of ATP is achieved!

6. REACTIONIn the next step of the Krebs cycle succinate is dehydrogenated (removal of two hydrogens and two electrons) to fumarate- this dehydrogenation reaction (oxidation of succinate) is catalyzed by the succinate dehydrogenase(SDH), an enzyme which is tightly associated with the inner mitochondrial membrane where it forms the crucial component of complex II of the mitochondrial electron transport chain;

- SDH is a large iron-sulfur protein (MW 100,000) which has a covalently bound FAD molecule as essential co-enzyme - the iron-sulfur (Fe-S) clusters of this interesting enzyme are known to somehow transport and feed electrons into the connected protein components of the electron transport chain

- this enzyme is competitively inhibited by the Krebs cycle blocker malonate, which played an important role in the unraveling of the intricate chemical reactions of the Krebs cycle

Succinate dehydrogenase, mitochondria & Aging theoriesAll multi-cellular biological organisms age for currently unknown reasons. Many theories have been brought forth in the past to explain the cause of biological aging. One of them is the free radical theory of aging. According to this currently popular theory, scientists believe that more and more free radicals and reactive oxygen species (ROS) accumulate in cells as living organisms age which eventually leads to progressive destruction of the integrity of proteins and to DNA mutations. As one of the suspected sources for these free radicals, a defective, “electron-leaking” electron transport chain has been postulated which may trickle more and more free radicals (unpaired electrons) into the cells. In the recent years - due to the fact that succinate dehydrogenase is an integral part of the complex II of the mitochondrial electron transport chain (ETC) and is an electron transporting Fe-S protein - scientists suspected this mitochondrial enzyme to be a weak link in the ETC. According to the popular “free radical theory of aging”, free electrons (= radicals) leaking from complex II of the ETC may lead to hydrogen peroxide formation, peroxidation processes, degradative oxidative stress in cells, cellular senescence … and – in the long haul – to the demise of the whole body (commonly referred to as aging).In the round worm Caenorhabditis elegans, scientists could show that mutations in the mev-1 gene (which codes for the beta-subunit of the succinate dehydrogenase enzyme = complex II of the mitochondrial ETC) leads to increased oxidative damage and a 37% shorter lifespan.Seems that the more food-derived electrons are "funneled" through the Krebs cycle and the mitochondrialelectron transport chain, the more free radicals might eventually be generated and the faster a cell might become damaged and go into cell senescence (= cell aging). Could it be that the more you eat the faster you age? Recent scientific findings strongly suggest that link.More and more scientific "dietary restriction" studies with roundworms, flies, and mice, clearly point to a strong connection of diet, energy input and aging. The less these animals were given to eat the older theygot.So, maybe we should think twice whether we want to go to this "all-you-can-eat" restaurant or havea free refill of this super-sized soda cup.

7. REACTION In the second last reaction, water is added to fumarate to form the 4-carbon molecule malate (or maleic acid).this hydration reaction is catalyzed by the enzyme fumarate hydratase

8.REACTION In the last reaction of the Krebs cycle, malate is converted into the 4-carbon molecule oxalate (= oxalic acid) is a typical dehydrogenation reaction (transfer of 2 hydrogens onto NAD+) involving the co-enzyme NAD+.- the oxidation of malate to oxalate is catalyzed by the enzyme malate dehydrogenase - this reaction is highly endergonic (ΔG0’ = +7.1 kcal/mole) and the equilibrium (under standard reaction conditions; 1 M, pH 7.0) is far to the left

- in intact cells, however, this chemical reaction proceeds towards the product side, since oxalate is rapidly removed by the following citrate synthase reaction in a “fully humming” Krebs cycle!- Oxalate, which concentration is extremely low within cells ( = 10-6 M) re-enters the Krebs cycle at step 1 and reacts with another delivered acetyl-CoA molecule to form citrate again; - the cycle is closed!

As stated previously all 8 steps of the Krebs cycle which are shown in the Graphic below occur in the matrix of the mitochondria

The Krebs cycle pays big energy dividend to the cell: the overall energy yield after degradation of 1 molecule of glucose is:

2 ATP, 6 NADH + H+ and 2 FADH2 molecules

which are retrieved after two complete rounds of the cycle- degradation of 1 molecule glucose during glycolysis in comparison brings the cell only 2 ATP and 2 NADH + H+

● The biggest energy profit the cell gains after ‘cashing-in’ the 6 NADH + H+ and 2 FADH2 molecules retrieved from the Krebs cycle at the earlier introduced electron transport chain (ETC) which is located

in the inner mitochondrial membrane

● There the ‘high-energy load’ of these two molecules is used in the cellular process called chemio-osmosis to synthesize ATP

1. Muchas de las enzimas del ciclo de Krebs son reguladas por retroalimentación negativa, por unión

alostérica del ATP, que es un producto de la vía y un indicador del nivel energético de la célula.

2. Entre estas enzimas, se incluye el complejo de la Piruvato Deshidrogenasa que sintetiza el Acetil-

CoA necesario para la primera reacción del ciclo a partir de piruvato, procedente de la glucólisis o

del catabolismo de aminoácidos.

3. También las enzimas citrato sintasa, isocitrato deshidrogenasa y α-Cetoglutarato

deshidrogenasa, que catalizan las tres primeras reacciones del ciclo de Krebs, son inhibidas por

altas concentraciones de ATP. Esta regulación frena este ciclo degradativo cuando el nivel

energético de la célula es bueno.

4. Algunas enzimas son también reguladas negativamente cuando el nivel de poder reductor de la

célula es elevado. El mecanismo que se realiza es una inhibición competitiva por producto (por

NADH) de las enzimas que emplean NAD+ como sustrato. Así se regulan, entre otros, los complejos

Piruvato Deshidrogenasa y Citrato Sintasa.

REGULACIÓN

Sitios de inhibición por parte de conservadores y que llevan a muerte celular