of glycolysis is known as the pay-off phase, characterised by a net gain of the energy-rich molecules atp and nadh. since glucose leads to two triose sugars in the preparatory phase, each reaction in the pay-off phase occurs twice per glucose molecule. this yields 2 nadh molecules and 4 atp molecules, leading to a net gain of 2 nadh molecules and 2 atp molecules from the glycolytic pathway per glucose. glyceraldehyde 3-phosphate (gadp) glyceraldehyde phosphate dehydrogenase (gapdh)an oxidoreductase d-1,3-bisphosphoglycerate (1,3bpg) nad+ + pi nadh + h+ the aldehyde groups of the triose sugars are oxidised, and inorganic phosphate is added to them, forming 1,3-bisphosphoglycerate. the hydrogen is used to reduce two molecules of nad+, a hydrogen carrier, to give nadh + h+ for each triose. hydrogen atom balance and charge balance are both maintained because the phosphate (pi) group actually exists in the form of a hydrogen phosphate anion (hpo2−4), which dissociates to contribute the extra h+ ion and gives a net charge of -3 on both sides. here, arsenate (3−), an anion akin to inorganic phosphate may replace phosphate as a substrate to form 1-arseno-3-phosphoglycerate. this, however, is unstable and readily hydrolyzes to form 3-phosphoglycerate, the intermediate in the next step of the pathway. as a consequence of bypassing this step, the molecule of atp generated from 1-3 bisphosphoglycerate in the next reaction will not be made, even though the reaction proceeds. as a result, arsenate is an uncoupler of glycolysis. 1,3-bisphosphoglycerate (1,3bpg) phosphoglycerate kinase (pgk)a transferase 3-phosphoglycerate (3pg) adp atp phosphoglycerate kinase (pgk) this step is the enzymatic transfer of a phosphate group from 1,3-bisphosphoglycerate to adp by phosphoglycerate kinase, forming atp and 3-phosphoglycerate. at this step, glycolysis has reached the break-even point: 2 molecules of atp were consumed, and 2 new molecules have now been synthesized. this step, one of the two substrate-level phosphorylation steps, requires adp; thus, when the cell has plenty of atp (and little adp), this reaction does not occur. because atp decays relatively quickly when it is not metabolized, this is an important regulatory point in the glycolytic pathway. adp actually exists as adpmg−, and atp as atpmg2−, balancing the charges at −5 both sides. cofactors: mg2+ 3-phosphoglycerate (3pg) phosphoglycerate mutase (pgm)a mutase 2-phosphoglycerate (2pg) phosphoglycerate mutase isomerises 3-phosphoglycerate into 2-phosphoglycerate. 2-phosphoglycerate (2pg) enolase (eno)a lyase phosphoenolpyruvate (pep) h2o enolase (eno) enolase next converts 2-phosphoglycerate to phosphoenolpyruvate. this reaction is an elimination reaction involving an e1cb mechanism. cofactors: 2 mg2+, one "conformational" ion to coordinate with the carboxylate group of the substrate, and one "catalytic" ion that participates in the dehydration. phosphoenolpyruvate (pep) pyruvate kinase (pk)a transferase pyruvate (pyr) adp + h+ atp a final substrate-level phosphorylation now forms a molecule of pyruvate and a molecule of atp by means of the enzyme pyruvate kinase. this serves as an additional regulatory step, similar to the phosphoglycerate kinase step. cofactors: mg2+ biochemical logic the existence of more than one point of regulation indicates that intermediates between those points enter and leave the glycolysis pathway by other processes. for example, in the first regulated step, hexokinase converts glucose into glucose-6-phosphate. instead of continuing through the glycolysis pathway, this intermediate can be converted into glucose storage molecules, such as glycogen or starch. the reverse reaction, breaking down, e.g., glycogen, produces mainly glucose-6-phosphate; very little free glucose is formed in the reaction. the glucose-6-phosphate so produced can enter glycolysis after the first control point. in the second regulated step (the third step of glycolysis), phosphofructokinase converts fructose-6-phosphate into fructose-1,6-bisphosphate, which then is converted into glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. the dihydroxyacetone phosphate can be removed from glycolysis by conversion into glycerol-3-phosphate, which can be used to form triglycerides. conversely, triglycerides can be broken down into fatty acids and glycerol; the latter, in turn, can be converted into dihydroxyacetone phosphate, which can enter glycolysis after the second control point. free energy changes concentrations of metabolites in erythrocytes: 584 compound concentration / mm glucose 5.0 glucose-6-phosphate 0.083 fructose-6-phosphate 0.014 fructose-1,6-bisphosphate 0.031 dihydroxyacetone phosphate 0.14 glyceraldehyde-3-phosphate 0.019 1,3-bisphosphoglycerate 0.001 2,3-bisphosphoglycerate 4.0 3-phosphoglycerate 0.12 2-phosphoglycerate 0.03 phosphoenolpyruvate 0.023 pyruvate 0.051 atp 1.85 adp 0.14 pi 1.0 the change in free energy, δg, for each step in the glycolysis pathway can be calculated using δg = δg°′ + rtln q, where q is the reaction quotient. this requires knowing the concentrations of the metabolites. all of these values are available for erythrocytes, with the exception of the concentrations of nad+ and nadh. the ratio of nad+ to nadh in the cytoplasm is approximately 1000, which makes the oxidation of glyceraldehyde-3-phosphate (step 6) more favourable. using the measured concentrations of each step, and the standard free energy changes, the actual free energy change can be calculated. (neglecting this is very common - the delta g of atp hydrolysis in cells is not the standard free energy change of atp hydrolysis quoted in textbooks). change in free energy for each step of glycolysis: 582–583 step reaction δg°′ (kj/mol) δg (kj/mol) 1 glucose + atp4− → glucose-6-phosphate2− + adp3− + h+ −16.7 −34 2 glucose-6-phosphate2− → fructose-6-phosphate2− 1.67 −2.9 3 fructose-6-phosphate2− + atp4− → fructose-1,6-bisphosphate4− + adp3− + h+ −14.2 −19 4 fructose-1,6-bisphosphate4− → dihydroxyacetone phosphate2− + glyceraldehyde-3-phosphate2− 23.9 −0.23 5 dihydroxyacetone phosphate2− → glyceraldehyde-3-phosphate2− 7.56 2.4 6 glyceraldehyde-3-phosphate2− + pi2− + nad+ → 1,3-bisphosphoglycerate4− + nadh + h+ 6.30 −1.29 7 1,3-bisphosphoglycerate4− + adp3− → 3-phosphoglycerate3− + atp4− −18.9 0.09 8 3-phosphoglycerate3− → 2-phosphoglycerate3− 4.4 0.83 9 2-phosphoglycerate3− → phosphoenolpyruvate3− + h2o 1.8 1.1 10 phosphoenolpyruvate3− + adp3− + h+ → pyruvate− + atp4− −31.7 −23.0 from measuring the physiological concentrations of metabolites in an erythrocyte it seems that about seven of the steps in glycolysis are in equilibrium for that cell type. three of the steps — the ones with large negative free energy changes — are not in equilibrium and are referred to as irreversible; such steps are often subject to regulation. step 5 in the figure is shown behind the other steps, because that step is a side-reaction that can decrease or increase the concentration of the intermediate glyceraldehyde-3-phosphate. that compound is converted to dihydroxyacetone phosphate by the enzyme triose phosphate isomerase, which is a catalytically perfect enzyme; its rate is so fast that the reaction can be assumed to be in equilibrium. the fact that δg is not zero indicates that the actual concentrations in the erythrocyte are not accurately known. regulation the enzymes that catalyse glycolysis are regulated via a range of biological mechanisms in order to control overall flux though the pathway. this is vital for both homeostatsis in a static environment, and metabolic adaptation to a changing environment or need. the details of regulation for some enzymes are highly conserved between species, whereas others vary widely. gene expression: firstly, the cellular concentrations of glycolytic enzymes are modulated via regulation of gene expression via transcription factors, with several glycolysis enzymes themselves acting as regulatory protein kinases in the nucleus. allosteric inhibition and activation by metabolites: in particular end-product inhibition of regulated enzymes by metabolites such as atp serves as negative feedback regulation of the pathway. allosteric inhibition and activation by protein-protein interactions (ppi). indeed, some proteins interact with and regulate multiple glycolytic enzymes. post-translational modification (ptm). in particular, phosphorylation and dephosphorylation is a key mechanism of regulation of pyruvate kinase in the liver. localization regulation by insulin in animals in animals, regulation of blood glucose levels by the pancreas in conjunction with the liver is a vital part of homeostasis. the beta cells in the pancreatic islets are sensitive to the blood glucose concentration. a rise in the blood glucose concentration causes them to release insulin into the blood, which has an effect particularly on the liver, but also on fat and muscle cells, causing these tissues to remove glucose from the blood. when the blood sugar falls the pancreatic beta cells cease insulin production, but, instead, stimulate the neighboring pancreatic alpha cells to release glucagon into the blood. this, in turn, causes the liver to release glucose into the blood by breaking down stored glycogen, and by means of gluconeogenesis. if the fall in the blood glucose level is particularly rapid or severe, other glucose sensors cause the release of epinephrine from the adrenal glands into the blood. this has the same action as glucagon on glucose metabolism, but its effect is more pronounced. in the liver glucagon and epinephrine cause the phosphorylation of the key, regulated enzymes of glycolysis, fatty acid synthesis, cholesterol synthesis, gluconeogenesis, and glycogenolysis. insulin has the opposite effect on these enzymes. the phosphorylation and dephosphorylation of these enzymes (ultimately in response to the glucose level in the blood) is the dominant manner by which these pathways are controlled in the liver, fat, and muscle cells. thus the phosphorylation of phosphofructokinase inhibits glycolysis, whereas its dephosphorylation through the action of insulin stimulates glycolysis. regulated enzymes in glycolysis the three regulatory enzymes are hexokinase (or glucokinase in the liver), phosphofructokinase, and pyruvate kinase. the flux through the glycolytic pathway is adjusted in response to conditions both inside and outside the cell. the internal factors that regulate glycolysis do so primarily to provide atp in adequate quantities for the cell's needs. the external factors act primarily on the liver, fat tissue, and muscles, which can remove large quantities of glucose from the blood after meals (thus preventing hyperglycemia by storing the excess glucose as fat or glycogen, depending on the tissue type). the liver is also capable of releasing glucose into the blood between meals, during fasting, and exercise thus preventing hypoglycemia by means of glycogenolysis and gluconeogenesis. these latter reactions coincide with the halting of glycolysis in the liver. in addition hexokinase and glucokinase act independently of the hormonal effects as controls at the entry points of glucose into the cells of different tissues. hexokinase responds to the glucose-6-phosphate (g6p) level in the cell, or, in the case of glucokinase, to the blood sugar level in the blood to impart entirely intracellular controls of the glycolytic pathway in different tissues (see below). when glucose has been converted into g6p by hexokinase or glucokinase, it can either be converted to glucose-1-phosphate (g1p) for conversion to glycogen, or it is alternatively converted by glycolysis to pyruvate, which enters the mitochondrion where it is converted into acetyl-coa and then into citrate. excess citrate is exported from the mitochondrion back into the cytosol, where atp citrate lyase regenerates acetyl-coa and oxaloacetate (oaa). the acetyl-coa is then used for fatty acid synthesis and cholesterol synthesis, two important ways of utilizing excess glucose when its concentration is high in blood. the regulated enzymes catalyzing these reactions perform these functions when they have been dephosphorylated through the action of insulin on the liver cells. between meals, during fasting, exercise or hypoglycemia, glucagon and epinephrine are released into the blood. this causes liver glycogen to be converted back to g6p, and then converted to glucose by the liver-specific enzyme glucose 6-phosphatase and released into the blood. glucagon and epinephrine also stimulate gluconeogenesis, which coverts non-carbohydrate substrates into g6p, which joins the g6p derived from glycogen, or substitutes for it when the liver glycogen store have been depleted. this is critical for brain function, since the brain utilizes glucose as an energy source under most conditions. the simultaneously phosphorylation of, particularly, phosphofructokinase, but also, to a certain extent pyruvate kinase, prevents glycolysis occurring at the same time as gluconeogenesis and glycogenolysis. hexokinase and glucokinase yeast hexokinase b (pdb: 1ig8​) all cells contain the enzyme hexokinase, which catalyzes the conversion of glucose that has entered the cell into glucose-6-phosphate (g6p). since the cell membrane is impervious to g6p, hexokinase essentially acts to transport glucose into the cells from which it can then no longer escape. hexokinase is inhibited by high levels of g6p in the cell. thus the rate of entry of glucose into cells partially depends on how fast g6p can be disposed of by glycolysis, and by glycogen synthesis (in the cells which store glycogen, namely liver and muscles). glucokinase, unlike hexokinase, is not inhibited by g6p. it occurs in liver cells, and will only phosphorylate the glucose entering the cell to form glucose-6-phosphate (g6p), when the glucose in the blood is abundant. this being the first step in the glycolytic pathway in the liver, it therefore imparts an additional layer