Glycolysis (Embden–Meyerhof pathway) is the sequence of reactions converting glucose (or glycogen) to pyruvate or lactate, with the production of ATP.
The first stage of cellular respiration, glycolysis, takes place in the cytoplasm of the cell. It is an anaerobic process and does not require oxygen.
A few eukaryotes (yeast and mature human red blood cells) and many prokaryotes (some bacteria) can survive on the energy produced by glycolysis alone.
Monosaccharide - the six carbon glucose molecule is the primary reactant of glycolysis.
Glycolysis is also the main pathway for metabolizing other dietary sugars, such as galactose and fructose.
The source of glucose may be from either carbohydrates or from glycogen (a molecule made of many glucose molecules) stored in muscle and liver cells.
Glucose enters cells that can not produce it (heterotrophic cells) in two ways:
- One method is through secondary active transport also known as coupled transport or co-transport. The transport takes place against the glucose concentration gradient.
- The second mechanism uses a group of integral proteins called GLUT proteins, also known as glucose transporter proteins. These transporters assist in the facilitated diffusion of glucose. They are encoded by the SLC2 genes. The human produces 14 GLUT proteins which are divided into 3 classes. Functioning of GLUT proteins 1 through 5 as glucose and/or fructose transporters are studied the most completely.
Mueckler M, Thorens B. The SLC2 (GLUT) family of membrane transporters. Mol Aspects Med. 2013;34(2-3):121-38. [pubmed]
10 steps of glycolysis pathway
Glucose is used to produce two 3-carbon pyruvate molecules.
Overall, glycolysis can be represented as:
C6H12O6 + 2NAD+ + 2Pi + 2ADP = 2Pyruvate + 2NADH + 2ATP
Nicotinamide adenine dinucleotide - NAD
Adenosine diphosphate - ADP
Adenosine triphosphate - ATP
To accomplish this process, 10 different enzymes are used.
Glycolysis has two main phases:
- Preparatory phase of glycolysis:
The preparatory phase of glycolysis pathway is the energy investment phase (endothermic) where the cell uses ATP.
- Pay off phase of glycolysis:
The last half of glycolysis is the energy payoff phase (exothermic) where ATP is produced.
Each of thease phases include 5 glycolytic pathway steps.
Preparatory phase of glycolysis pathway (the endothermic activation phase)
In order for glycolysis to begin, activation energy, from an ATP molecule, must be provided.
Step 1: Glucose to Glucose-6-phosphate (Hexokinase)
The first reaction of 10 glycolysis steps - substrate-level phosphorylation is catalyzed by hexokinase.
Hexokinase is one of three enzymes involved in regulation of glycolysis.
One ATP is used to phosphorylate glucose to form glucose-6-phosphate.
This step prevents the phosphorylated glucose molecule from leaving the cell because the negatively charged hydrophilic phosphate does not allow to cross the hydrophobic interior of the plasma membrane.
Trapped inside a cell in the form of glucose 6-phosphate, glucose has three metabolic options.
For example, glucose can be oxidized via glycolysis for the primary purpose of ATP production, stored as glycogen, or oxidized in the pentose phosphate pathway to generate NADPH and ribose for nucleic acid synthesis.
Three possible metabolic pathway of glucose metabolism: glycolysis, the pentose phosphate pathway, and glycogen synthesis
Step 2: Glucose-6-phosphate to Fructose-6-phosphate (Phosphohexose isomerase)
In the second step, glucose-6-phosphate molecule is then rearranged by an isomerase to form fructose-6-phosphate.
Step 3: Fructose-6-phosphate to Fructose-1,6-diphosphate (Phosphofructokinase-1)
At this point, another ATP molecule must phosphorylate the fructose-6-phosphate, producing fructose-1,6-diphosphate.
The third step of glycolysis, the phosphorylation, is catalyzed by the enzyme phosphofructokinase.
Phosphofructokinase is a rate-limiting enzyme.
- It is active when the concentration of ADP is high;
- It is less active when ADP levels are low and the concentration of ATP is high.
Step 4: Fructose-1,6-diphosphate to Glyceraldehyde-3-phosphate and Dihydroxyacetone phosphate (Aldolase)
The fourth step of glycolysis utilizes an enzyme, aldolase, to split fructose-1,6-bisphosphate into one molecule of glyceraldehyde-3-phosphate (PGAL, G3P) and one molecule of dihydroxyacetone phosphate (DHAP).
Only glyceraldehyde-3-phosphate can continue through the rest of the reactions.
Step 5: Dihydroxyacetone phosphate to Glyceraldehyde-3-phosphate (Triose-phosphate isomerase)
In the fifth step, dihydroxyacetone phosphate (DHAP), which is an isomer of glyceraldehyde-3-phosphate (PGAL), is converted by an isomerase into a second molecule of PGAL.
This is the energy investment portion of the glycolysis pathway where two ATP molecules have been used.
These glyceraldehyde-3-phosphates act as the reactants for pay off phase of glycolysis.
Pay off phase of glycolysis pathway (the exothermic phase)
Pay off phase of glycolysis is a sequence of exothermic reactions that provides energy for the cell.
Step 6: Glyceraldehyde-3-phosphate to 1,3-Bisphosphoglycerate (Glyceraldehyde 3-phosphate dehydrogenase)
Following preparatory phase of glycolysis, in the sixth step, each glyceraldehyde-3-phosphate (PGAL) is oxidized.
The transfer of electrons and a proton to the carrier molecule NAD+ (nicotinamide adenine dinucleotide) creates NADH.
This is the point where the process of glycolysis can slow down or stop. The availability of the oxidized form of the electron carrier, NAD+, is a limiting factor. To keep this step going NADH must be oxidized back into NAD+ without intermission. If oxygen is available in the system, the NADH will be oxidized in the final phase of cellular respiration - the stage of oxidative phosphorylation.
Without oxygen, an alternate pathway (fermentation) can provide the oxidation of NADH to NAD+.
The oxidized form of glyceraldehyde-3-phosphate is now able to attract a free phosphate ion in the cytosol, forming 1,3-biphosphoglycerate (PGAP).
Step 7: 1,3-Biphosphoglycerate to 3-Phosphoglycerate (Phosphoglycerate kinase)
Phosphoglycerate kinase catalyzes the seventh step of glycolysis.
Following the formation of 1,3-biphosphoglycerate (PGAP), two ADP molecules each remove one phosphate group from each PGAP to form 3-phosphoglycerate (PGA) and ATP.
At this point the net change in the number of ATP molecules is zero.
Next, the two 3-phosphoglycerate (PGA) molecules are each oxidized, forming two water molecules and two phosphoenolpyruvate (PEP) molecules.
Step 8: 3-Phosphoglycerate to 2-Phosphoglycerate (Phosphoglycerate mutase)
In the eighth step, moving the phosphate group in 3-phosphoglycerate from the third carbon to the second carbon, an isomerase creates 2-phosphoglycerate.
Step 9: 2-Phosphoglycerate to Phosphoenolpyruvate (Enolase)
The ninth glycolysis step is a dehydration reaction. Enolase forces 2-phosphoglycerate to lose water and produces phosphoenolpyruvate (PEP).
The rearrangement of some of the electrons causes the remaining phosphate group to become unstable.
Step 10: Phosphoenolpyruvate to Pyruvate (Pyruvate kinase)
Finally, in tenth step catalyzed by the pyruvate kinase, two ADP molecules each remove the remaining phosphate group from each phosphoenolpyruvate (PEP) molecule.
The result products of glycolysis are four ATP molecules, two NADH molecules and two pyruvate molecules.
Two ATP molecules are used in the first half of the pathway in the preparatory phase of glycolysis.
The entire glycolysis process, including preparatory and pay off phases of glycolysis, produces a net gain of two ATP molecules.
Pyruvate can be further oxidized in mitochondria.
Intermediates and products of glycolysis as substrates for other metabolic pathways
Dihydroxyacetone phosphate (DHAP)
Adipose and liver tissues which have a high power for triacylglycerol synthesis contain glycerol 3-phosphate dehydrogenase.
The dehydrogenase converts dihydroxyacetone phosphate (DHAP) into glycerol 3-phosphate, which is a important substrate in the pathways of triglyceride and phospholipid synthesis:
dihydroxyacetonephosphate + NADH + H+ ↔ glycerol 3-phosphate + NAD+
Red blood cells use 1,3-bisphosphoglycerate (1,3-BPG), an intermediate in pay off phase of glycolysis pathway, to generate 2,3-bisphosphoglycerate (2,3-BPG), which is an allosteric regulator of the interaction of oxygen with hemoglobin.
2,3-BPG is present in human red blood cells and binds with greater affinity to deoxygenated hemoglobin (near respiring tissue) than to oxygenated hemoglobin (in the lungs). It functions to stabilize the low oxygen affinity state of the oxygen carrier and make it more likely for oxygen to be released to neighboring tissues.
The interconversion of these two bisphosphoglycerates is catalyzed by bisphosphoglycerate mutase:
1,3-bisphophoglycerate ↔ 2,3-bisphosphoglycerate
Pyruvate, the end product of glycolysis, can receive an amino group by transamination and produce the amino acid alanine.
Irreversible steps of glycolysis include three enzymes, each of which catalyzes a reaction which involved in regulation of this pathway: hexokinase, phosphofructokinase-1, and pyruvate kinase.
Hexokinase refers to the first reaction of 10 glycolysis steps;
phosphofructokinase-1 to the third glycolysis step;
pyruvate kinase to the tenth step.
Hexokinase – the enzyme of the first irreversible step of glycolysis
Phosphorylation of glucose serves to activate the sugar for metabolism.
As the concentration of glucose 6-phosphate increases it reaches the point where it inhibits hexokinase. This is an example of product inhibition.
If hepatocytes had no other glucose-trapping enzyme than hexokinase, the liver would soon stop extracting glucose from the blood and the body would experience hyperglycemia. The problem is solved by the induction of the enzyme glucokinase by insulin.
Glucokinase is not inhibited by glucose 6-phosphate. The function of glucokinase is to trap glucose when the blood glucose concentration rises after a meal.
Phosphofructokinase-1 - the enzyme of the second rate limiting step of glycolysis
Phosphofructokinase-1 (PFK-1) is the major enzyme participating in regulation of glycolysis.
The energy needs of the cell and hormonal signaling by insulin and glucagon regulate performance phosphofructokinase-1 (PFK-1). It is inhibited by adenosine triphosphate (ATP) and citrate, and activated by adenosine monophosphate (AMP) and fructose 2,6-bisphosphate (Fru-2,6-P2).
Citrate is an intermediate in the mitochondrial TCA cycle. When there are excess of ATPs in the cell, ATPs inhibit mitochondrial isocitrate dehydrogenase, the key regulatory enzyme of the TCA cycle, resulting in an accumulation of citrate and slowing the flux of glucose through glycolysis.
Fructose 2,6-bisphosphate (Fru-2,6-P2) is an allosteric activator of phosphofructokinase-1 (PFK-I) and its concentration depends on the insulin/glucagon ratio.
The bifunctional enzyme phosphofructokinase 2fructose-2,6-bisphosphatase (PFK-2/FBPase-2) control the level fructose 2,6-bisphosphate directly by synthesizing and breaking it down.
The activities of this enzyme are regulated differently in different tissues.
In liver cells the PFK-2/FBPase-2 enzyme is regulated through phosphorlyation/dephosphorylation reactions.
Glucagon activates adenylyl cyclase and raises the cAMP level of the cell. cAMP-activated protein kinase phosphorylates PFK-2/FBPase-2 enzyme what activates fructose-2,6-bisphosphatase. FBPase-2 dephosphorylates fructose 2,6-bisphosphate causing its level in the cell to decline and producing fructose 6-phosphate and Pi.
Decreased the fructose 2,6-bisphosphate level decreases phosphofructokinase-1 activity, and ultimately slowes down the flux of glucose through glycolysis.
Binding of insulin to its receptor activates a phosphoprotein phosphatase that dephosphorylates PFK-2/FBPase-2 enzyme. This process allows the PFK-2 portion of the enzyme to phosphorylate fructose 6-phosphate using ATP and thereby causes the F2,6P2 concentration to rise and to increase the flux of glucose through glycolysis.
In skeletal muscle cells, the PFK-2/FBPase-2 enzyme can not be activated by cAMP-activated protein kinase A. As a result, the PFK-2/FBPase-2 enzyme constitutively synthesizes fructose 2,6-bisphosphate and glycolysis is not inhibited by epinephrine-induced intracellular signaling.
In heart muscle, an isoenzyme of PFK-2/FBPase-2 enzyme has multiple phosphorylation sites including one phosphorylated by AMP-activated protein kinase (5' adenosine monophosphate-activated protein kinase or AMPK). Energy depletion results in a high AMP/ATP ratio which stimulates activation of AMPK. In turn, activated AMPK phosphorylates phosphofructokinase 2 (PFK-2), which increases the intracellular concentration of fructose 2,6-bisphosphate, and thereby a rate of glycolysis and energy production.
Pyruvate kinase – the last regulatory step in glycolysis pathway
The last step of glycolysis is inhibited by ATP and activated by fructose-l,6-bisphosphate.
The activity of the liver isoenzyme of pyruvate kinase is also regulated by phosphorylation and dephosphorylation. Glucagon stimulates cAMP synthesis in liver cells (hepatocytes), causing cAMP-activated protein kinase A to phosphorylate pyruvate kinase. The phosphorylated form of protein kinase is inactive.
Conditions with especially active glycolysis
In the fed state when the body is actively processing nutrients.
Increased postprandial blood glucose levels stimulate sectection of insulin by the cells of the pancreas. Insulin, in turn, stimulates glucose metabolism in muscle, liver, and fat cells, but not in the brain.
Two sources of glucose for muscle are localized glycogen stores within the muscle and glucose extracted from the blood.
At rest muscle cells derive most of their energy from the oxidation of fatty acids.
Exercising muscle oxidizes glucose as well as fatty acids. More and more of the energy in muscle will be received from glucose as the intensity of exercise increases. In hard exercising muscle, the demand for oxygen can outrun the oxygen supply so that the muscle has to rely only on glycolysis to satisfy its ATP needs. As a result, under anaerobic condition, the muscle increases lactate production which serves as the substrate of gluconeogenesis in the liver.
Lactate is the result of the reduction of pyruvate by NADH. The enzyme lactatede hydrogenase utilizes the NADH obtained from the sixth step of glycolysis. The formation of lactate allows the regeneration of NAD+ so that glycolysis continues even in the absence of oxygen to supply ATP.
Cancer cells consume glucose at a much higher rate and produce much more lactic acid than normal cells. The increased ATP produced by glycolysis in cancer cells is used for fatty acid, protein, and DNA synthesis.
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