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Glycolysis, TCA, Electron Transport, ATP Synthesis

Content : - Glycolysis - Substrate level phosphorylation - Fermentation - Mitochondria - Tricarboxylic acid cycle - Electron Transport Chain - ATP formation - fat metabolism

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Cellular energy is generated through oxidation reactions. Substrates for such oxidations are mainly carbohydrates and fats. But first carbohydrates have to be hydrolyzed to sugars and fats to fatty acids. The cellular oxidation reactions proceed in several stages , that means the compounds are not immediately oxidized to CO2 and H2. The first stage in the oxidation of glucose is called glycolysis and proceeds in the cytosol , which is the fluid section of the cytoplasm . A simplified scheme of the glycolysis pathway is shown below.

Even though the pathway will generate energy , it initially consumes 2 ATP. Glucose is phosphorylated to glucose-6-phosphate, which then it is converted to fructose-6-phosphate and phosphorylated again to fructose-1,6-diphosphate. All the following reactions yield energy after this initial energy consumption (in form of ATP). In the next step the six carbon compound (fructose-1,6-diphosphate) is cleaved into 2 identical three-carbon compounds (glyceraldehyde-3-phosphate). In the next step glyceraldehyde-3-phosphate is oxidized to 1,3-bisphosphoglycerate: the O=C-H group is oxidized to a O=C-O-PO3-- group. The two electrons released in this process have enough energy to reduce NAD+ to NADH. 1,3-diphosphoglycerate itself is a high energy compound and it can convert ADP to ATP by providing the energy necessary (G= +7.3 kcal/mol ). The 3-phosphoglycerate generated in this reaction is transformed into phosphoenol pyruvate. Phosphoenol pyruvate is another high energy compound, it can give its phosphate to ADP to form ATP, while it is converted to pyruvate. This is the last step in the glycolysis pathway.
In the first sequence 2 ATP are consumed, in the latter sequence 2 ATP are generated per 1 molecule of glyceraldehyde-3-phosphate. Since there are 2 molecules glyceraldehyde-3-phosphates, 4 ATP are generated . Thus, the total amount of ATP generated in glycolysis is 2 ATP per one molecule of glucose. The formation of ATP from ADP in glycolysis is called substrate level phosphorylation since the phosphate is transferred from a substrate (e.g. phosphoenol pyruvate) to ADP
All reactions in the glycolysis are catalyzed by enzymes suspended in the cytosol . Hexokinase and phosphofructokinase, which catalyze the two initial phosphorylations are the most important ones, because they control the pathway with their activity . Phosphofructokinase is inhibited by high ATP concentrations, thus the pathway stops. When ATP levels are low the enzyme functions again and ATP can be produced . activity of phosphofructokinase

The endproduct of glycolysis is pyruvate. If oxygen is present then pyruvate is oxidized to CO2. This process takes place in the mitochondrion and involves another pathway called tricarboxylic acid cycle. If oxygen is limited or absent pyruvate is reduced to lactic acid using NADH which was produced during glycolysis .

If we consider the reactions from glucose to lactate, no net oxidation has occurred, since the electrons obtained in the reaction from 3-phosphoglyceraldehyde to 1,3 diphosphoglycerate have been returned to pyruvate when it was reduced to lactate. The reaction from pyruvate to lactate is called fermentation. If you do vigorous exercises then the body’s demand for ATP is very high. Oxygen can not be carried fast enough into the muscle cells to oxidize all the pyruvate, so some of it is converted to lactate or lactic acid. This is cause for sour muscles.
Lactate fermentation is not the only type of fermentation. For example yeast cell convert pyruvate into ethanol:

In presence of oxygen the complete oxidation of pyruvate takes place in the mitochondria.    size comparison     micrograph     Image

size comparison     micrograph     Image The mitochondrion is an organelle with an inner and an outer membrane. The outer membrane contains pores (called porines) through which most small and medium sized molecules can pass unhindered. The inner membrane has infolds, the space inside these infolds is called matrix. It contains a colloidal liquid very much like the cytosol . The inner membrane is not freely permeable, like the plasma membrane it has transporter molecules which regulate the flux of substances in and out of the matrix. The space enclosed by the infolds is called cristae and the inner membrane in these locations contains a series of enzymes called the electron transport chain. Also located in this section of the inner membrane are proteins called ATPase, which are the site of ATP synthesis.

Tricarboxylic acid cycle (TCA)
Pyruvate enters the matrix of the mitochondria via a transporter molecule. Once inside it takes part in a sequence of reactions called the tricarboxylic acid cycle or Krebs cycle after its discoverer. In the first step pyruvate reacts with Coenzyme A to form acetyl CoA . Electrons released in this process are accepted by NAD+ to form NADH . Acetyl CoA is the form in which the carbon from glycolysis enters the tricarboxylic acid cycle

The TCA is basically a sequence of reactions in which pyruvate is oxidized to CO2. The electrons generated in these oxidation reactions are used to reduce a total of 3 NAD+ to 3 NADH and 1 FAD to FADH2. In addition 1 ATP is generated.

The first reaction in TCA is the addition of acetyl CoA to oxaloacetate to form citrate. Oxaloacetate is a compound with 4 carbon atoms, acetyl CoA has 2 carbons, thus citrate must have 6 carbons. In the TCA citrate is eventually transformed back to oxaloacetate , the two carbon atoms are lost in form of CO2. Now the cycle can start all over with another acetyl CoA. The first reaction of oxaloacetate to citrate is catalyzed by the enzyme citratesynthase. This enzyme is inhibited by high NADH concentration. We will see later how this can regulate the pathway.

Electron Transport Chain (TCA)
Now, we have generated all this NADH and FADH2 in the matrix of the mitochondrion, how can this reducing energy be converted into ATP? (remember the major function of the mitochondria is the formation of ATP). The schematic diagram below illustrates the processes that lead to ATP formation.

(image )The mechanism involves enzymes located in the infolded sections ( cristae ) of the mitochondrial inner membrane, the so called enzymes of the electron transport chain. Some of the enzymes, the cytochromes are able to accept and donate elctrons. Cytochromes contain an iron binding structural subunit, called heme , which is directly involved in the electron transfer. There are other enzymes present in the electron transport chain, which are also capable of either accepting or donating electrons. These enzymes are organized in four complexes. Complex I can accept electrons from NADH only, whereas complex II accepts electrons only from FADH2. Complex III in turn will accept electrons from complex I or II via a mobile electron carrier called CoQ (coenzyme Q). Complex III will give the electrons to complex IV via another mobile carrier called cytochrome c. In a final pass the electrons are given to O2 which is being reduced to H2O.
While the electrons are handed down from complex to complex they can carry out work. This is illustrated below

The actual mechanism may be somewhat different then the one shown here, but it is a good model for what happens. The electrons are released from NADH and are passed on to complex I. They are high energy electrons and after being accepted by an iron-protein in complex I the reduced iorn ( Fe++) causes this complex to change its conformation. (Even though two electrons are released from 1 NADH, we just consider one electron being added to Fe+++ to form Fe++) Image that a proton pump is part of this complex I. When the iron protein is in its oxidized state (Fe+++) the configuration of the pump allows a proton to be attached from the matrix side (drawing 1). After the iron protein has accepted the high energy electrons from NADH, the reduced iron ( Fe++) causes a conformational change in the iron protein, which in turn makes the proton pump to flip over to the cristae side and release the proton (drawing 2). The pump can return to its orginal state, if the reduced iron protein gives the electrons to the next electron carrier. Then Fe++gives up an electron and returns to Fe+++, consequently the original configuration is established (drawing 3). The electrons are passed from complex to complex and each time they will cause the transfer of protons from the matrix into the cristae. After having been passed to complex IV the electrons lost most of their energy, however, they still have enough energy to be passed to oxygen which is reduced to water.

As mentioned above NADH releases its two electrons to complex I, where a total of 4 protons are transported from the matrix to the cristae. The same two electrons can then transport 4 more protons via complex III and 2 protons via complex VI. Thus a total of 10 protons are moved by one NADH. The electrons of FADH2 have less energy, i.e. FADH2 has less reducing power than NADH . The electrons of FADH2 are given to complex II and then are passed to complex III via the mobile electron carrier CoQ. After that they pass to complex IV and to oxygen. As you can see only 6 protons are moved by one FADH2.

ATP formation: A strong proton gradient builds up in the cristae (pH=5). In addition to the enzymes of the electron transport chain, a transporter protein called ATPase (or ATP synthase) is also located in the membrane. It allows protons to pass from the high concentration side (cristae, pH=5) to the matrix were the pH= 7. The energy released in this transport is used by the transporter to catalyze the reaction of ADP + Pi = ATP. The ATP can then travel out of the mitochondria (via a transporter) into the cytoplasm or into organelles were it is needed. The mechanism by which ATP is formed is called oxidative phosphorylation, since the energy for the phosphorylation of ADP to ATP is derived from oxidation reactions

Fat metabolism: We recall that acetyl CoA initiated the entire reaction sequence which is also known as respiration . Acetyl CoA was oxidized in the TCA, the electrons from this oxidation were passed along the electron transport chain, where protons were moved across the membrane and the resulting gradient drove ATP synthesis. Glucose is not the only source for acetyl CoA. It is also formed in the degradation of fatty acids coming from fats. Glucose yields only 2 acetyl CoA per glucose molecule , whereas typical fats will generated about 27 acetyl CoA per molecule fat. Thus, one molecule fat can generate much more ATP than glucose. However, fat metabolism quite complex : fats have to be transported from fat cell tissue to metabolizing cells in form of vesicles, which are taken up through endocytosis and degraded into fatty acids, which then have to be converted into acetyl CoA in a process called beta oxidation . The pathway of glucose to pyruvate to acetyl CoA is much simpler and preferred by the cell as principal source for acetyl CoA.

The fat cells in our bodies (called adipose tissue) contain most of our fat reserves. There is another type of fat cells which constitutes only 2 - 3 % of al fat cells. They are called brown fat cells. The mitochondria of these cells do not contain ATPase but a protein called thermogenine which functions as a proton channel. It allows the protons to pass from the cristae into the matrix, however the released energy is not used to make ATP, but it is dissipated in form of heat. Thus, fat energy is turned into heat. (This is incidentally the way our bodies can generate heat). Some individuals have only a very small content of brown fat cells. The thermogenine transport molecule is stimulated by norepinephrine, but also by nicotine and caffeine. In obese individuals this mechanism may not function.