of control of the glycolytic pathway in this organ. phosphofructokinase bacillus stearothermophilus phosphofructokinase (pdb: 6pfk​) phosphofructokinase is an important control point in the glycolytic pathway, since it is one of the irreversible steps and has key allosteric effectors, amp and fructose 2,6-bisphosphate (f2,6bp). fructose 2,6-bisphosphate (f2,6bp) is a very potent activator of phosphofructokinase (pfk-1) that is synthesized when f6p is phosphorylated by a second phosphofructokinase (pfk2). in the liver, when blood sugar is low and glucagon elevates camp, pfk2 is phosphorylated by protein kinase a. the phosphorylation inactivates pfk2, and another domain on this protein becomes active as fructose bisphosphatase-2, which converts f2,6bp back to f6p. both glucagon and epinephrine cause high levels of camp in the liver. the result of lower levels of liver fructose-2,6-bisphosphate is a decrease in activity of phosphofructokinase and an increase in activity of fructose 1,6-bisphosphatase, so that gluconeogenesis (in essence, "glycolysis in reverse") is favored. this is consistent with the role of the liver in such situations, since the response of the liver to these hormones is to release glucose to the blood. atp competes with amp for the allosteric effector site on the pfk enzyme. atp concentrations in cells are much higher than those of amp, typically 100-fold higher, but the concentration of atp does not change more than about 10% under physiological conditions, whereas a 10% drop in atp results in a 6-fold increase in amp. thus, the relevance of atp as an allosteric effector is questionable. an increase in amp is a consequence of a decrease in energy charge in the cell. citrate inhibits phosphofructokinase when tested in vitro by enhancing the inhibitory effect of atp. however, it is doubtful that this is a meaningful effect in vivo, because citrate in the cytosol is utilized mainly for conversion to acetyl-coa for fatty acid and cholesterol synthesis. tigar, a p53 induced enzyme, is responsible for the regulation of phosphofructokinase and acts to protect against oxidative stress. tigar is a single enzyme with dual function that regulates f2,6bp. it can behave as a phosphatase (fructuose-2,6-bisphosphatase) which cleaves the phosphate at carbon-2 producing f6p. it can also behave as a kinase (pfk2) adding a phosphate onto carbon-2 of f6p which produces f2,6bp. in humans, the tigar protein is encoded by c12orf5 gene. the tigar enzyme will hinder the forward progression of glycolysis, by creating a build up of fructose-6-phosphate (f6p) which is isomerized into glucose-6-phosphate (g6p). the accumulation of g6p will shunt carbons into the pentose phosphate pathway. pyruvate kinase yeast pyruvate kinase (pdb: 1a3w​) the final step of glycolysis is catalysed by pyruvate kinase to form pyruvate and another atp. it is regulated by a range of different transcriptional, covalent and non-covalent regulation mechanisms, which can vary widely in different tissues. for example, in the liver, pyruvate kinase is regulated based on glucose availability. during fasting (no glucose available), glucagon activates protein kinase a which phosphorylates pyruvate kinase to inhibit it. an increase in blood sugar leads to secretion of insulin, which activates protein phosphatase 1, leading to dephosphorylation and re-activation of pyruvate kinase. these controls prevent pyruvate kinase from being active at the same time as the enzymes that catalyze the reverse reaction (pyruvate carboxylase and phosphoenolpyruvate carboxykinase), preventing a futile cycle. conversely, the isoform of pyruvate kinasein found in muscle is not affected by protein kinase a (which is activated by adrenaline in that tissue), so that glycolysis remains active in muscles even during fasting. post-glycolysis processes the overall process of glycolysis is: glucose + 2 nad+ + 2 adp + 2 pi → 2 pyruvate + 2 nadh + 2 h+ + 2 atp if glycolysis were to continue indefinitely, all of the nad+ would be used up, and glycolysis would stop. to allow glycolysis to continue, organisms must be able to oxidize nadh back to nad+. how this is performed depends on which external electron acceptor is available. anoxic regeneration of nad+ one method of doing this is to simply have the pyruvate do the oxidation; in this process, pyruvate is converted to lactate (the conjugate base of lactic acid) in a process called lactic acid fermentation: pyruvate + nadh + h+ → lactate + nad+ this process occurs in the bacteria involved in making yogurt (the lactic acid causes the milk to curdle). this process also occurs in animals under hypoxic (or partially anaerobic) conditions, found, for example, in overworked muscles that are starved of oxygen. in many tissues, this is a cellular last resort for energy; most animal tissue cannot tolerate anaerobic conditions for an extended period of time. some organisms, such as yeast, convert nadh back to nad+ in a process called ethanol fermentation. in this process, the pyruvate is converted first to acetaldehyde and carbon dioxide, and then to ethanol. lactic acid fermentation and ethanol fermentation can occur in the absence of oxygen. this anaerobic fermentation allows many single-cell organisms to use glycolysis as their only energy source. anoxic regeneration of nad+ is only an effective means of energy production during short, intense exercise in vertebrates, for a period ranging from 10 seconds to 2 minutes during a maximal effort in humans. (at lower exercise intensities it can sustain muscle activity in diving animals, such as seals, whales and other aquatic vertebrates, for very much longer periods of time.) under these conditions nad+ is replenished by nadh donating its electrons to pyruvate to form lactate. this produces 2 atp molecules per glucose molecule, or about 5% of glucose's energy potential (38 atp molecules in bacteria). but the speed at which atp is produced in this manner is about 100 times that of oxidative phosphorylation. the ph in the cytoplasm quickly drops when hydrogen ions accumulate in the muscle, eventually inhibiting the enzymes involved in glycolysis. the burning sensation in muscles during hard exercise can be attributed to the release of hydrogen ions during the shift to glucose fermentation from glucose oxidation to carbon dioxide and water, when aerobic metabolism can no longer keep pace with the energy demands of the muscles. these hydrogen ions form a part of lactic acid. the body falls back on this less efficient but faster method of producing atp under low oxygen conditions. this is thought to have been the primary means of energy production in earlier organisms before oxygen reached high concentrations in the atmosphere between 2000 and 2500 million years ago, and thus would represent a more ancient form of energy production than the aerobic replenishment of nad+ in cells. the liver in mammals gets rid of this excess lactate by transforming it back into pyruvate under aerobic conditions; see cori cycle. fermentation of pyruvate to lactate is sometimes also called "anaerobic glycolysis", however, glycolysis ends with the production of pyruvate regardless of the presence or absence of oxygen. in the above two examples of fermentation, nadh is oxidized by transferring two electrons to pyruvate. however, anaerobic bacteria use a wide variety of compounds as the terminal electron acceptors in cellular respiration: nitrogenous compounds, such as nitrates and nitrites; sulfur compounds, such as sulfates, sulfites, sulfur dioxide, and elemental sulfur; carbon dioxide; iron compounds; manganese compounds; cobalt compounds; and uranium compounds. aerobic regeneration of nad+, and disposal of pyruvate in aerobic eukaryotes, a complex mechanism has developed to use the oxygen in air as the final electron acceptor. aerobic prokaryotes, which lack mitochondria, use a variety of simpler mechanisms. firstly, the nadh + h+ generated by glycolysis has to be transferred to the mitochondrion to be oxidized, and thus to regenerate the nad+ necessary for glycolysis to continue. however the inner mitochondrial membrane is impermeable to nadh and nad+. use is therefore made of two “shuttles” to transport the electrons from nadh across the mitochondrial membrane. they are the malate-aspartate shuttle and the glycerol phosphate shuttle. in the former the electrons from nadh are transferred to cytosolic oxaloacetate to form malate. the malate then traverses the inner mitochondrial membrane into the mitochondrial matrix, where it is reoxidized by nad+ forming intra-mitochondrial oxaloacetate and nadh. the oxaloacetate is then re-cycled to the cytosol via its conversion to aspartate which is readily transported out of the mitochondrion. in the glycerol phosphate shuttle electrons from cytosolic nadh are transferred to dihydroxyacetone to form glycerol-3-phosphate which readily traverses the outer mitochondrial membrane. glycerol-3-phosphate is then reoxidized to dihydroxyacetone, donating its electrons to fad instead of nad+. this reaction takes place on the inner mitochondrial membrane, allowing fadh2 to donate its electrons directly to coenzyme q (ubiquinone) which is part of the electron transport chain which ultimately transfers electrons to molecular oxygen o2, with the formation of water, and the release of energy eventually captured in the form of atp. the glycolytic end-product, pyruvate (plus nad+) is converted to acetyl-coa, co2 and nadh + h+ within the mitochondria in a process called pyruvate decarboxylation. the resulting acetyl-coa enters the citric acid cycle (or krebs cycle), where the acetyl group of the acetyl-coa is converted into carbon dioxide by two decarboxylation reactions with the formation of yet more intra-mitochondrial nadh + h+. the intra-mitochondrial nadh + h+ is oxidized to nad+ by the electron transport chain, using oxygen as the final electron acceptor to form water. the energy released during this process is used to create a hydrogen ion (or proton) gradient across the inner membrane of the mitochondrion. finally, the proton gradient is used to produce about 2.5 atp for every nadh + h+ oxidized in a process called oxidative phosphorylation. conversion of carbohydrates into fatty acids and cholesterol the pyruvate produced by glycolysis is an important intermediary in the conversion of carbohydrates into fatty acids and cholesterol. this occurs via the conversion of pyruvate into acetyl-coa in the mitochondrion. however, this acetyl coa needs to be transported into cytosol where the synthesis of fatty acids and cholesterol occurs. this cannot occur directly. to obtain cytosolic acetyl-coa, citrate (produced by the condensation of acetyl coa with oxaloacetate) is removed from the citric acid cycle and carried across the inner mitochondrial membrane into the cytosol. there it is cleaved by atp citrate lyase into acetyl-coa and oxaloacetate. the oxaloacetate is returned to mitochondrion as malate (and then back into oxaloacetate to transfer more acetyl-coa out of 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 can be used as is, as a structural component of cellular membranes, or it can be used to synthesize the steroid hormones, bile salts, and vitamin d. conversion of pyruvate into oxaloacetate for the citric acid cycle pyruvate molecules produced by glycolysis are actively transported across the inner mitochondrial membrane, and into the matrix where they can either be oxidized and combined with coenzyme a to form co2, acetyl-coa, and nadh, or they can be carboxylated (by pyruvate carboxylase) to form oxaloacetate. this latter reaction "fills up" the amount of oxaloacetate in the citric acid cycle, and is therefore an anaplerotic reaction (from the greek meaning to "fill up"), increasing the cycle's capacity to metabolize acetyl-coa when the tissue's energy needs (e.g. in heart and skeletal muscle) are suddenly increased by activity. in the citric acid cycle all the intermediates (e.g. citrate, iso-citrate, alpha-ketoglutarate, succinate, fumarate, malate and oxaloacetate) are regenerated during each turn of the cycle. adding more of any of these intermediates to the mitochondrion therefore means that that additional amount is retained within the cycle, increasing all the other intermediates as one is converted into the other. hence the addition of oxaloacetate greatly increases the amounts of all the citric acid intermediates, thereby increasing the cycle's capacity to metabolize acetyl coa, converting its acetate component into co2 and water, with the release of enough energy to form 11 atp and 1 gtp molecule for each additional molecule of acetyl coa that combines with oxaloacetate in the cycle. to cataplerotically remove oxaloacetate from the citric cycle, malate can be transported from the mitochondrion into the cytoplasm, decreasing the amount of oxaloacetate that can be regenerated. furthermore, citric acid intermediates are constantly used to form a variety of substances such as the purines, pyrimidines and porphyrins. intermediates for