CELLULAR RESPIRATION Applied Medical Chemistry II Course

Aim of the lecture This lecture discuss the steps of cellular respiration and its regulation

Maye M. Ragab

Super Visor / Dr. Neveen Introduction individual enzymic reactions were analyzed in an effort to explain the mechanisms of catalysis. However, in cells, these reactions rarely occur in isolation, but rather are organized into multistep sequences called pathways). In a pathway, the product of one reaction serves as the substrate of the subsequent reaction. Different pathways can also intersect, forming an integrated and purposeful network of chemical reactions. These are collectively called metabolism, which is the sum of all the chemical changes occurring in a cell, a tissue, or the body. Most pathways can be classified as either catabolic (degradative) or anabolic (synthetic). Anabolic pathways • form complex end products from simple precursors, for example, the synthesis of the polysaccharide, glycogen, from glucose. • Anabolic reactions require energy (are endergonic), which is generally provided by the breakdown of ATP to (ADP) and inorganic phosphate (Pi). • Anabolic reactions often involve chemical reductions in which the reducing power is most frequently provided by the electron donor NADPH . Catabolic reactions • break down complex molecules, such as proteins, polysaccharides, and lipids, to a few simple molecules, for example, CO2, NH3 (ammonia), and water. • Catabolic reactions serve to capture chemical energy in the form of (ATP) from the degradation of energy-rich fuel molecules. Energy generation by degradation of complex molecules occurs in three stages as in the following figure :

[Note: Catabolic pathways are typically oxidative, and require coenzymes such as NAD+.] [Note: Pathways that regenerate a component are called cycles.]

Overview Of Glycolysis : • The glycolytic pathway is employed by all tissues for the breakdown of glucose to provide energy (in the form of ATP) and intermediates for other metabolic pathways. • Glycolysis is at the hub of carbohydrate metabolism because virtually all sugars— whether arising from the diet or from catabolic reactions in the body can ultimately be converted to glucose. • Pyruvate is the end product of glycolysis in cells with mitochondria and an adequate supply of oxygen. This series of ten reactions is called aerobic glycolysis because oxygen is required to reoxidize the NADH formed during the oxidation of glyceraldehyde3-phosphate . Aerobic glycolysis sets the stage for the oxidative decarboxylation of pyruvate to acetyl CoA, a major fuel of the TCA (or citric acid) cycle.

• Alternatively, pyruvate is reduced to lactate as NADH is oxidized to NAD+ . This conversion of glucose to lactate is called anaerobic glycolysis because it can occur without the participation of oxygen. Anaerobic glycolysis allows the production of ATP in : ✓ tissues that lack mitochondria (for example, red blood cells) ✓ or in cells deprived of sufficient oxygen.

Reduction of pyruvate to lactate • Lactate, formed by the action of lactate dehydrogenase, is the final product of anaerobic glycolysis in eukaryotic cells. • (the reason for lactate formation ) is In exercising skeletal muscle, NADH production exceeds the oxidative capacity of the respiratory chain, This results in an elevated NADH/NAD+ ratio, favoring reduction of pyruvate to lactate. Therefore, during intense exercise, lactate accumulates in muscle, causing a drop in the intracellular pH, potentially resulting in cramps. • Much of this lactate eventually diffuses into the bloodstream, and can be used by the liver to make glucose.

• What happened in Glycolysis ?

The conversion of glucose to pyruvate occurs in two stages : 1. The first five reactions of glycolysis correspond to an energy investment phase in which the phosphorylated forms of intermediates are synthesized at the expense of ATP.

2. The subsequent reactions of glycolysis constitute an energy generation phase in which a net of two molecules of ATP are formed by substrate-level phosphorylation .

Energy yield from glycolysis

1. Anaerobic glycolysis: • Two molecules of ATP are generated for each molecule of glucose converted to two molecules of lactate. • There is no net production or consumption of NADH.

2. Aerobic glycolysis: • The direct consumption and formation of ATP is the same as in anaerobic glycolysis that is, a net gain of two ATP per molecule of glucose. • Two molecules of NADH are also produced per molecule of glucose. • Ongoing aerobic glycolysis requires the oxidation of most of this NADH by the electron transport chain, producing approximately three ATP for each NADH molecule entering the chain.

5 regulation of glycolysis • The regulation of glycolysis by allosteric activation or inhibition, or the phosphorylation/dephosphorylation of rate-limiting enzymes, is short term that is, they influence glucose consumption over periods of minutes or hours. • There are Four regulatory enzymes of glycolysis (Hexokinase ,Glucokinase , phosphofructokinase and pyruvate kinase).

✓ Lets start first with the Phosphorylation of glucose ( the first step in glycolysis )

• Phosphorylated sugar molecules do not readily penetrate cell membranes because there are no specific transmembrane carriers for these compounds. • and because they are too polar to diffuse through the lipid core of membranes. The irreversible phosphorylation of glucose therefore, effectively traps the sugar as cytosolic glucose 6-phosphate, thus committing it to further metabolism in the cell. • Mammals have several isozymes of the enzyme hexokinase that catalyze the phosphorylation of glucose to glucose 6-phosphate:

1. Hexokinase: In most tissues, the phosphorylation of glucose is catalyzed by hexokinase, one of three regulatory enzymes of glycolysis. - Hexokinase is inhibited by the reaction product, glucose 6-phosphate, which accumulates when further metabolism of this hexose phosphate is reduced. Hexokinase has a low Km (and, therefore, a high affinity for glucose. - This permits the efficient phosphorylation and subsequent metabolism of glucose even when tissue concentrations of glucose are low . - Hexokinase, however, has a low Vmax for glucose and, therefore, cannot sequester (trap) cellular phosphate in the form of phosphorylated hexoses, or phosphorylate more sugars than the cell can use.

2. Glucokinase: In liver parenchymal cells and β cells of the pancreas,glucokinase is the predominant enzyme responsible for the phosphorylation of glucose. In β cells,glucokinase functions as the glucose sensor,determining the threshold for insulin secretion . - Glucokinase differs from hexokinase in several important properties. For example, it has a much higher Km, requiring a higher glucose concentration for half-saturation. - Thus, glucokinase functions only when the intracellular concentration of glucose in the hepatocyte is elevated, such as during the brief period following consumption of 6

a carbohydrate-rich meal, when high levels of glucose are delivered to the liver via the portal vein. - Glucokinase has a high Vmax, allowing the liver to effectively remove the flood of glucose delivered by the portal blood. This prevents large amounts of glucose from entering the systemic circulation following a carbohydrate rich meal.

Regulation by fructose 6-phosphate and glucose: Glucokinase activity is not directly inhibited by glucose 6-phosphate as are the other hexokinases, but rather is indirectly inhibited by fructose 6- phosphate (which is in equilibrium with glucose 6-phosphate, a product of glucokinase), and is indirectly stimulated by glucose (a substrate of glucokinase) via the following mechanism:

Glucokinase regulatory protein (GKRP) in the liver regulates the activity of glucokinase through reversible binding. In the presence of fructose 6- phosphate, glucokinase is translocated into the nucleus and binds tightly to the regulatory protein, thus rendering the enzyme inactive .

3- phospho fructokinase : The irreversible phosphorylation reaction catalyzed by phospho fructokinase) Phosphofructokinase (PFK) is the enzyme that controls the third step of glycolysis, the conversion of fructose-6-phosphate (F6P) into fructose-1,6-biphosphate (F1,6BP). It works by transferring a phosphate group from ATP to F6P. This is the slowest reaction in glycolysis and therefore is the rate-limiting step. PFK-1 is controlled by the available concentrations of the substrates ATP and fructose2,6-bisphosphate. - PFK-1 is inhibited allosterically by elevated levels of ATP and high level of citrate (produced from krebs cycle ). - PFK-1 is activated allosterically by high concentrations of AMP or Fructose 2,6- bisphosphate .

4- Pyruvate kinase : The conversion of PEP to pyruvate is catalyzed by pyruvate kinase, the third irreversible reaction of glycolysis. This step increases the concentration of ATP in the cell. - high ATP levels inhibit PK. Thus, pyruvate kinase is subject to negative feedback when glycolysis is running too hot. - In liver, pyruvate kinase is activated by fructose 1,6-bisphosphate, the product of the phosphofructo kinase reaction and this called forward stimulation .

✓ Superimposed on these moment-to-moment effects are slower, and often more profound, hormonal influences on the amount of enzyme protein synthesized. These effects can result in 10-fold to 20- fold increases in enzyme activity that typically occur over hours to days.

• Although the current focus is on glycolysis, reciprocal changes occur in the rate-limiting enzymes of gluconeogenesis. • Regular consumption of meals rich in carbohydrate or administration of insulin initiates an increase in the amount of glucokinase, phosphofructokinase, and pyruvate kinase in liver, These changes reflect an increase in gene transcription, resulting in increased enzyme synthesis. • High activity of these three enzymes favors the conversion of glucose to pyruvate • Conversely, gene transcription and synthesis of glucokinase, phosphofructokinase, and pyruvate kinase are decreased when plasma glucagon is high and insulin is low, for example, as seen in fasting or diabetes. • And vice versa in acse of

Overview Of TriCarboxylic Acid Cycle • TCA cycle, also called the Krebs cycle or the citric acid cycle . • It is the final pathway where the oxidative metabolism of carbohydrates, amino acids, and fatty acids converge, their carbon skeletons being converted to CO2. This oxidation provides energy for the production of the majority of ATP in most animals. • Pyruvate, the end product of aerobic glycolysis, must be transported into the mitochondrion of eukaryotes or the cytosol of prokaryotes before it can enter the TCA cycle. This is accomplished by a specific pyruvate transporter that helps pyruvate cross the inner mitochondrial membrane. Once in the matrix, pyruvate is converted to acetyl CoA by the pyruvate dehydrogenase complex.

the entry of one acetyl CoA into one round of the TCA cycle does not lead to the net production or consumption of intermediates. [Note: Two carbons entering the cycle as acetyl CoA are balanced by two CO2 exiting.]

• TCA cycle is followed by the electron transport chain process , which oxidize the reduced coenzymes produced by the cycle.

• The TCA cycle is an aerobic pathway, because O2 is required as the final electron acceptor.

Functions of TCA cycle : 1. Conversion of Acetyl CoA to Co2 and H2O. 2. Produces NADH and FADH that lead to Formation of ATP as a result of oxidative phosphorylation . 3. Produces GTP as a result of substrate level phosphorylation . 4. Provides for synthetic reactions ,for example conversion of amino acids to glucose

• What happened in this cycle ? Oxaloacetate is first condensed with an acetyl group from acetyl coenzyme A (CoA), to form citrate (a tricarboxylic acid) and then is regenerated as the cycle is completed.

** this figure shows the main important products beyond the intermediates that lead to releasing energy by electron transport chain

Energy Produced By TCA cycle : • Two carbon atoms enter the cycle as acetyl CoA and leave as CO2. • The cycle does not involve net consumption or production of oxaloacetate or of any other intermediate. • Four pairs of electrons are transferred during one turn of the cycle: ✓ three pairs of electrons reducing three NAD+ to NADH and one pair reducing FAD to FADH2. ✓ Oxidation of one NADH by the electron transport chain leads to formation of approximately three ATP. ✓ whereas oxidation of FADH2 yields approximately two ATP . ✓ and one GTP equivalent to one ATP . ✓ The total yield of ATP from the Krebs cycle are 24 ATP .

Regulation of TCA cycle :

Pre Regulation : • I means here a regulation before the process begin , as we know that TCA cycle is proceeded by a transition step in which Pyruvate transformed into Acetyl CoA by Pyruvate dehydrogenase complex . • This complex is composed of aggregate of three enzymes ( PDH or E1,E2 and E3 ). • In addition to the enzymes participating in the conversion of pyruvate to acetyl CoA, the complex also contains two tightly bound regulatory enzymes, pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase. • Covalent modification by the two regulatory enzymes that are part of the complex alternately activate and inactivate E1 (PDH). • The cyclic AMP-independent PDH kinase phosphorylates and, thereby, inhibits E1, whereas PDH phosphatase dephosphorylates and activates E1). • The kinase itself is allosterically activated by ATP, acetyl CoA, and NADH. Therefore, in the presence of these high-energy signals, the PDH complex is turned off. • Pyruvate is a potent inhibitor of PDH kinase. Therefore, if pyruvate concentrations are elevated, E1 will be maximally active. • Calcium is a strong activator of PDH phosphatase, stimulating E1 activity. This is particularly important in skeletal muscle, where release of Ca2+ during contraction stimulates the PDH complex, and thereby energy production. [Note: Although covalent regulation by the kinase and phosphatase is key, the complex is also subject to product (NADH, acetyl CoA) inhibition.]

Post Regulation : • here I mean the regulation of the cycle itself by its intermediates. • the TCA cycle is controlled by the regulation of several enzyme activities . • The most important of these regulated enzymes are those that catalyze reactions with highly negative ΔG0: citrate synthase, isocitrate dehydrogenase, and α- ketoglutarate dehydrogenase complex. • Reducing equivalents needed for oxidative phosphorylation are generated by the pyruvate dehydrogenase complex and the TCA cycle, and both processes are upregulated in response to a rise in ADP.

To Sum Up see the opposite figure :

Overview of Electron Transport Chain Energy-rich molecules, such as glucose, are metabolized by a series of oxidation reactions ultimately yielding CO2 and water . The metabolic intermediates of these reactions donate electrons to specific coenzymes (NAD+) and (FAD) to form the energy-rich reduced coenzymes, NADH and FADH2. These reduced coenzymes can, in turn, each donate a pair of electrons to a specialized set of electron carriers, collectively called the electron transport chain. As electrons are passed down the electron transport chain, they lose much of their free energy. Part of this energy can be captured and stored by the production of ATP from ADP and inorganic phosphate (Pi), This process is called oxidative phosphorylation , The remainder of the free energy not trapped as ATP is used to drive ancillary reactions such as Ca2+ transport into mitochondria, and to generate heat. What happened in Electron Transport chain : The components of the electron transport chain are located in the inner membrane. Although the outer membrane contains special pores, making it freely permeable to most ions and small molecules, the inner mitochondrial membrane is a specialized structure that is impermeable to most small ions, including H+, Na+, and K+, and small molecules such as ATP, ADP, pyruvate, and other metabolites important to mitochondrial function . the matrix contains NAD+ and FAD (the oxidized forms of the two coenzymes that are required as hydrogen acceptors) and ADP and Pi, which are used to produce ATP. The inner mitochondrial membrane can be disrupted into five separate protein complexes, called Complexes I, II, III, IV, and V. • I–IV each contain part of the electron transport chain. Each complex accepts or donates electrons to relatively mobile electron carriers, such as coenzyme Q and cytochrome c. Each carrier in the electron transport chain can receive electrons from an electron donor, and can subsequently donate electrons to the next carrier in the chain. The electrons ultimately combine with oxygen and protons to form water. • Complex V is a protein complex that contains a domain (Fo) that spans the inner mitochondrial membrane, and a domain (F1) that appears as a sphere that protrudes into the mitochondrial matrix. Complex V catalyzes ATP synthesis and so is referred to as ATP synthase.

Brief steps of Electron transport chain reaction :

Important Note : The net energy resulted of one glucose break down in aerobic cell respiration are 32 ATP . Glycolysis : 2 ATP Pyruvate decarboxylation : 6ATP Krebs cycle : 24 ATP Then 2+6+24 = 32 16

For Illustration Only from page (17-19) Some detailed information about the mechanism of action of electron transport chain : With the exception of coenzyme Q, all members of this chain are proteins.These may function as enzymes as is the case with the dehydrogenases, may contain iron as part of an iron–sulfur center, may be coordinated with a porphyrin ring as in the cytochromes, or may contain copper as does the cytochrome a + a3 complex.

NADH dehydrogenase: Both electrons but only one proton (that is, a hydride ion, :H–) are transferred to the NAD+, forming NADH plus a free proton, H+. The free proton plus the hydride ion carried by NADH are next transferred to NADH dehydrogenase, a protein complex (Complex I) embedded in the inner mitochondrial membrane. Complex I has a tightly bound molecule of flavin mono nucleotide (FMN, a coenzyme, that accepts the two hydrogen atoms (2e– + 2H+), becoming FMNH2. NADH dehydrogenase also contains iron atoms paired with sulfur atoms to make iron–sulfur centers. These are necessary for the transfer of the hydrogen atoms to the next member of the chain, coenzyme Q. coenzyme Q :is a mobile carrier and can accept hydrogen atoms both from FMNH2, produced on NADH dehydrogenase (Complex I), and from FADH2, produced on succinate dehydrogenase (Complex II).

CoQ transfers electrons to Complex III. CoQ, then, links the flavoproteins to the cytochromes. Cytochromes: The remaining members of the electron transport chain are cytochromes. Each contains a heme group (a porphyrin ring plus iron). Unlike the heme groups of hemoglobin, the cytochrome iron is reversibly converted from its ferric (Fe3+) to its ferrous (Fe2+) form as a normal part of its function as a reversible carrier of electrons. Electrons are passed along the chain from CoQ to cytochromes bc1 (Complex III), c, and a + a3 (Complex IV) Cytochrome a + a3: This cytochrome complex is the only electron carrier in which the heme iron has an available coordination site that can react directly with O2, and so also is called cytochrome oxidase. At this site, the transported electrons, O2, and free protons are brought together, and O2 is reduced to water. Overview of Oxidative Phosphorylation : The transfer of electrons down the electron transport chain is energetically favored because NADH is a strong electron donor and molecular oxygen is an avid electron acceptor. However, the flow of electrons from NADH to oxygen does not directly result in ATP synthesis.

1. Proton pump: Electron transport is coupled to the phosphorylation of ADP by the transport (“pumping”) of protons (H+) across the inner mitochondrial membrane from the matrix to the intermembrane space at Complexes I, III, and IV. This process creates an electrical gradient (with more positive charges on the outside of the membrane than on the inside) and a pH gradient (the outside of the membrane is at a lower pH than the inside. The energy generated by this proton gradient is sufficient to drive ATP synthesis. Thus, the proton gradient serves as the common intermediate that couples oxidation to phosphorylation. 2. ATP synthase: The enzyme complex ATP synthase (Complex V) synthesizes ATP using the energy of the proton gradient generated by the electron transport chain. after protons have been pumped to the cytosolic side of the inner mitochondrial membrane, they reenter the matrix by passing through a channel in the membrane- spanning domain (Fo ) of Complex V, driving rotation of Fo and, at the same time, dissipating the pH and electrical gradients. Fo rotation causes conformational changes in the extra-membranous F1 domain that allow it to bind ADP + Pi, phosphorylate ADP to ATP, and release ATP.

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