Glycolysis is a process that involves:
Glucose + 2 NAD+ + 2 ADP + 2 Pi → 2 pyruvate + 2 NADH + 2 H+ + 2 ATP
Glycolysis could not continue indefinitely if all NAD+ was used up and glycolysis would cease. Organisms must be capable of oxidizing NADH back into NAD+ in order to allow glycolysis to continue. The external electron acceptor will determine how this is done.
1. Anoxic regeneration of NAD+
This can be done by simply allowing the pyruvate to oxidize; during this process, the pyruvate becomes lactate (the conjugate basis of lactic acids) in a process known as lactic acid fermentation.
Pyruvate + NADH + H+ → lactate + NAD+
This happens in yogurt bacteria (the lactic acid is what causes the milk curdle). Hypoxic or partially anaerobic conditions can also cause this process. This is common, for instance, when muscles are overworked and starved of oxygen. This is the cellular last resort for energy in many tissues; animal tissue can’t tolerate prolonged periods of anaerobic conditions.
In a process known as ethanol fermentation, some organisms like yeast convert NADH back into NAD+. This process converts the pyruvate first to acetaldehyde, carbon dioxide, then to ethanol.
In the absence of oxygen, lactic acid fermentation and alcohol fermentation can both occur. Many single-cell organisms can use glycolysis to get their energy from this anaerobic fermentation.
Anoxic recovery of NAD+ can only be used to produce energy during intense exercise in vertebrates for a short time, and it is limited to a maximum effort of 10 seconds to 2 mins. It can sustain muscle activity in divers, like seals, whales, and other aquatic vertebrates for much longer periods of times at lower intensities. These conditions allow NADH to replenish NAD+ by donating electrons to pyruvate in order to form lactate.
This results in 2 ATP molecules per glucosemolecule, or approximately 5% of glucose’s energy potential (38ATP molecules in bacteria). This method produces ATP at a rate of about 100 times faster than oxidative phosphorylation. When hydrogen ions build up in muscle cells, the pH of the cytoplasm drops quickly. This eventually leads to the inhibition of enzymes involved with glycolysis.
The release of hydrogen ions from glucose oxidation to carbon dioxide, water and glucose fermentation can cause muscle burning during exercise. This happens when the aerobic metabolism is unable to keep up with the energy needs of the muscles. These hydrogen ions are a part lactic acid.
This less efficient, but more effective method of producing ATP in low oxygen conditions is what the body uses. This method is believed to have been the main source of energy production for organisms prior to oxygen reaching high levels in the atmosphere. It would be a more ancient form than aerobic replenishment of NAD+ cells.
Mammals’ livers eliminate excess lactate and convert it into pyruvate in aerobic conditions. See Cori cycle.
Anaerobic glycolysis is the fermentation of pyruvate into lactate. However, the end of glycolysis is the production of pyruvate regardless if oxygen is present.
The above examples of fermentation show that NADH is oxidized through the transfer of two electrons to Pyruvate. Anaerobic bacteria can use many compounds as terminal electron acceptors for cellular respiration. These include nitrogenous compounds such as nitrates or nitrites, as well as sulfur compounds such as sulfites and sulfur dioxide.
2. Aerobic regeneration of NAD+, and disposal of pyruvate
Aerobic organisms have a complex mechanism that uses oxygen in the air as the final electron acceptor.
- First, glycolysis generates NADH + H+ that must be transferred to mitochondria to be oxidized and regenerated with NAD+. The inner mitochondrial membrane is not permeable to NADH or NAD+. Two “shuttles” are used to move electrons from NADH across mitochondrial membrane. These are the malate/aspartate shuttle, and the glycerol/phosphate shuttle. The former transfers electrons from NADH to cytosolic Oxaloacetate, which forms malate. The malate traverses the inner mitochondrial cell membrane to the mitochondrial matrix where it is reoxidized with NAD+, forming intra-mitochondrial Oxaloacetate or NADH. The oxaloacetate can then be re-cycled into the cytosol by being converted to aspartate, which is easily transported out of the mitochondrial membrane. Glycerol phosphate shuttle electrons are transferred from cytosolic NADH to dihydroxyacetone. This forms glycerol-3 phosphate, which is easily traverses the outer mitochondrial cell membrane. Glycerol-3 phosphate is then reoxidized into dihydroxyacetone, which donates its electrons instead of NAD+. This happens on the inner mitochondrial membrane. FADH2 then donates its electrons to coenzymeQ (ubiquinone), which is part the electron transport chain that ultimately transfers electrons into molecular oxygen (O2) and finally releases energy in the form ATP.
- In a process known as pyruvate degradation, the glycolytic end product, pyruvate, plus NAD+, is converted within mitochondria to acetyl–CoA, CO2 or NADH + H+.
- The acetyl CoA formed is then entered into the citric acid cycle or Krebs Cycle, where the acetyl groups of the acetyl CoA are converted to carbon dioxide through two decarboxylation reaction with the formation yet more intra-mitochondrial NAD + H+.
- The electron transport chain uses oxygen as an electron acceptor to make water. This energy is used to create an inner membrane hydrogen ion or proton gradient.
- The proton gradient can be used to produce approximately 2.5 ATP per NADH + H+ oxidized, a process known as oxidative phosphorylation.
3. Conversion of carbohydrates into fatty acids and cholesterol
Glycolysis produces pyruvate, which is an intermediary in the conversions of carbohydrates to fatty acids and cholesterol. This is achieved by the conversion of pyruvate to acetyl CoA in the mitochondrion. This acetyl CoA must be transported to the cytosol for the synthesis and maintenance of cholesterol and fatty acids. This cannot happen directly. Citrate, which is formed by the condensation of Acetyl CoA and oxaloacetate, is used to obtain cytosolic-acetyl CoA. It is carried through the inner mitochondrial membrane into cytosol.
It is then cleaved into acetylcoA and oxaloacetate by ATP citrate lyase. The oxaloacetate goes back to the mitochondrion as a malate, and then back into oxaloacetate in order to release more acetyl CoA from the mitochondrion. The cytosolic acetyl-CoA can be carboxylated by acetyl-CoA carboxylase into malonyl CoA, the first committed step in the synthesis of fatty acids, or it can be combined with acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) which is the rate limiting step controlling the synthesis of cholesterol. Cholesterol is a structural component in cellular membranes and can be used for vitamin D, steroid hormones, and bile salts.
4. Conversion of pyruvate into oxaloacetate for the citric acid cycle
The pyruvate molecules that are produced by glycolysis are transported actively across the inner mitochondrial membrane and into the matrix, where they can be either oxidized and mixed with coenzyme to form CO2, acetyl CoA and NADH or carboxylated (by the pyruvate carboxylase to form oxaloacetate). This reaction “fills up” the citric acid cycle with oxaloacetate and is called an anaplerotic response (from the Greek meaning “fill up”), which increases the cycle’s ability to metabolize Acetyl-CoA as the tissue’s energy requirements (e.g. Activity is suddenly increased in the heart and skeletal muscles. All intermediates in the citric acid cycle (e.g. Each turn of the cycle results in the regeneration of citrate, fumarate and succinate as well as alpha-ketoglutarate, succinate and fumarate.
Any intermediate added to the mitochondrion increases the number of intermediates, which in turn means more intermediates are retained. The addition of oxaloacetate significantly increases the amount of all citric acid intermediates. This allows the cycle to metabolize more acetyl CoA and converts its acetate component to CO2. With enough energy, the cycle can also form 11 ATP molecules and 1 GTP molecule each additional molecule acetyl CoA which is combined with oxaloacetate.
Malate can be transported from a mitochondrion to the cytoplasm in order to cataplerotically eliminate oxaloacetate (from the citric cycle). This decreases the amount of oxaloacetate which can be regenerated. Citric acid intermediates can be used to make a wide range of substances, including the porphyrins, pyrimidines, and purines.