Glycolysis

Definitions & Key takeaways

Glycolysis is a process that breaks down glucose into two molecules of pyruvate. Pyruvate is then used in the citric acid cycle to produce energy in the form of ATP. Glycolysis occurs in the cytoplasm of cells and does not require oxygen.

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Let’s say that you just ate a big slice of pizza with onions, mushrooms, bell peppers, and jalapenos. To pull energy out of the glucose in that pizza or really any food, requires glycolysis.
Glycolysis is a series of enzymatic reactions in which glucose, a 6 carbon sugar molecule, is broken down into two 3 carbon pyruvate molecules.
And as glucose gets processed, energy is produced in the form of adenosine triphosphate, or ATP. Now, glycolysis happens in the cytoplasm of cells, and no special organelles or even oxygen are needed to turn glucose into ATP.
Therefore, all cells can use glucose to make energy; and it’s possible to do glycolysis even when oxygen levels are low.
Glycolysis can be divided into two phases: an energy-consuming phase, and an energy-producing phase. It’s like a business investment - the cell needs to spend some energy before it can start making energy, and like any good investment the cell gets more energy back than it puts in.
The energy-consuming phase requires ATP, and the energy-producing phase generates ATP, as well as other molecules like reduced nicotinamide adenine dinucleotide, or NADH, which can be used to make ATP.
We can keep track of all of this using an energy counter. Going back to that delicious pizza, first, glucose from those ingredients has to first get from the small intestine into the bloodstream.
In response to high blood glucose, the pancreatic beta-cells secrete insulin. Now, to get inside the cells, glucose utilizes glucose transporters, or GLUT, which are on the cell membrane.
In fact, some GLUTs like GLUT2 in the liver and pancreatic beta-cells are particularly responsive to glucose in the presence of insulin.
Once glucose gets inside the cell, it’s prevented from diffusing across the cell membrane back into the circulation by enzymes called kinases which phosphorylate the glucose.
Adding a phosphate group changes the shape of the glucose molecule, which means it can’t easily diffuse out of the cell, a bit like a criminal that’s handcuffed to the table in an interrogation room.
The phosphate comes from the breakdown of ATP into ADP and phosphate - so this initial phosphorylation step drops us to -1 on that energy counter.
Specifically, there are two enzymes called hexokinase and glucokinase, and they both add a phosphate group to the 6th carbon in the glucose molecule, turning it into glucose-6-phosphate.
Both enzymes pretty much do the same thing, but hexokinase is found in all cells, whereas glucokinase, like GLUT2, is induced by the presence of insulin, and is found in the liver cells and the beta-cells of the pancreas.
This first step is irreversible, meaning that the reaction can only go in the glucose to glucose-6-phosphate direction, and not vice versa.
Glucose-6-phosphate is converted to its isomer, fructose-6-phosphate by an enzyme called phosphoglucoisomerase. So at this point, it’s still a 6 carbon molecule.
Fructose-6-phosphate is then phosphorylated by the enzyme phosphofructokinase-1, or PFK1, which adds a phosphate group to the 1st carbon on the fructose molecule, making fructose-1,6-bisphosphate.
This is the second irreversible reaction in glycolysis and it also uses ATP as a phosphate source - so we’re at -2 on that counter now.
This reaction is considered the rate-limiting step of glycolysis - meaning that how fast PFK1 converts fructose-6-phosphate to fructose-1,6-bisphosphate determines the speed at which all of glycolysis happens.
In other words, it’s the rate limiting step of glycolysis. It’s a bit like an assembly line in a factory, if the slowest step is putting tires on a car, then that’s the step that determines how many cars get built in a day.
Because of this, cells closely regulate PFK1 activity by using another enzyme, called phosphofructokinase 2 - or PFK2. You see - PFK2 can also phosphorylate fructose-6-phosphate - but it adds phosphate to the 2nd carbon instead, making fructose 2,6-bisphosphate.
PFK2 activity varies depending on the level of glucose in the blood. When the body is well-fed, like right after eating that slice of pizza, blood glucose levels go up, and the pancreas secretes insulin, which activates PFK2 - resulting in more fructose-2,6 bisphosphate.
Now, here’s the key - increased levels of fructose-2,6 bisphosphate activates PFK1, which means it increases the rate of available PFK1 enzymes.
So more PFK1 means that the slowest step in glycolysis speeds up, and more glucose is turned into energy. More tires, more cars.
Now, when the body is in a fasting state, like a few hours after a meal, blood glucose goes back down, and the pancreas secretes glucagon instead of insulin.
Glucagon inhibits PFK2, resulting in less fructose-2,6-bisphosphate, which inhibits PFK1, decreasing the rate of PFK1 enzymes, and that slows down glycolysis.
Fewer tires, fewer cars. PFK1 is also inhibited in other ways.
For example, when cells are in high energy states, there is a lot of ATP floating around as well as citrate, because that’s a by product of fatty acid synthesis.
Both ATP and citrate inhibit PFK1, because cells that have lots of energy don’t need to generate even more. Now when cells do need energy, PFK1 becomes very active in generating fructose 1,6 bisphosphate.
Fructose 1,6 bisphosphate is cleaved by the enzyme aldolase into two 3 carbon molecules, glyceraldehyde-3-phosphate, or G3P, and dihydroacetone-phosphate, or DHAP.
Only G3P can go down the glycolysis pathway, so an isomerase enzyme converts DHAP into G3P. As a result, for each glucose molecule, there are two G3P molecules.
Each G3P is converted into 1,3 bisphosphoglycerate, or 1,3-BPG, by an enzyme called G3P-dehydrogenase. G3P-dehydrogenase has 2 roles, it removes a hydrogen from G3P and gives it to a nearby NAD+ molecule, making NADH as a byproduct.
It also adds a phosphate group to the 1st carbon of G3P, making 1,3-BPG. Now since there are two G3P molecules, this happens twice, resulting in two NADH molecules.
Each NADH molecule enters the electron transport chain in the mitochondria and makes roughly 3 ATP. An enzyme called phosphoglycerate kinase, removes a phosphate from the 1st carbon of 1,3-BPG and gives it to ADP, making 3-phosphoglycerate, and ATP as a byproduct.
So we’ll add two ATPs to our counter because this reaction happens twice - so we’re back at 0. Next, an enzyme called a mutase moves the phosphate on 3-phosphoglycerate to the 2nd carbon, making 2-phosphoglycerate.
After that, an enzyme called enolase removes a water molecule from 2 phosphoglycerate and makes phosphoenolpyruvate - or PEP.
Finally, the enzyme pyruvate kinase transfers a phosphate from PEP to ADP, making pyruvate, and ATP as a byproduct. This is our 3rd and last irreversible reaction of glycolysis, and again we’ll add 2 ATPs to our counter because this reaction happens twice.
As it turns out, pyruvate kinase is also regulated by the cell. Interestingly, fructose-1,6 bisphosphate upregulates pyruvate kinase - a process called feed-forward regulation, because it’s sort of like one enzyme priming another one because it’s clear that things are about to get busy.
On the other hand, high levels of ATP and the amino acid alanine downregulate pyruvate kinase activity. Alanine comes from skeletal muscle breakdown when fasting, and it’s used as a substrate for making new glucose.
So high levels of alanine signify that the body needs to make new glucose, not break it down in glycolysis. Once pyruvate is made, glycolysis is pretty much over.
Until this point, the process has worked without the need of oxygen, so glycolysis is anaerobic. And to this point, we’ve generated a total of two ATPs in this process.
So although this is a good investment, it’s not a great one. Fortunately, most cells have mitochondria, and have access to oxygen - because that’s where the payoff really becomes obvious.
That’s because pyruvate can enter the mitochondria and participate in the Krebs cycle, also called the citric acid cycle, and the electron transport chain to make more ATP.
And in the end, after all the mitochondrial reactions, you’ll end up with a net total of roughly 30 to 32 ATPs. Some cell don’t have access to sufficient oxygen; like an exercising skeletal muscle cell, or a red blood cell that lacks mitochondria.
In those situations, the cell can use the enzyme lactate dehydrogenase to remove hydrogen from an NADH molecule and give it to pyruvate, creating lactate, and NAD+ as a byproduct.
NAD+ is crucial because it’s needed to work with G3P-dehydrogenase and keep glycolysis going. Now, normally, lactate is removed from our blood by the kidneys.
However, if local lactate levels rise too quickly, it can sometimes build-up, and it’s responsible for some of the muscle soreness you develop when you exercise.
Alright, a quick recap. Glycolysis breaks down a 6-carbon glucose molecule into two 3-carbon pyruvate molecules, without the use of any organelles or oxygen.
Overall there’s a net production of 2 ATP and 2 NADH molecules, which in the mitochondria make roughly 3 ATPs per NADH.