Biochemistry-metabolism
Glucose-6-phosphate is converted to its isomer, fructose-6-phosphate by an enzyme called
Glycolysis
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.
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.
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Biochemistry-metabolism
Now since there are two G3P molecules, this happens twice, resulting in two NADH molecules. Each NADH Summary
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 Alright, a quick recap. Glycolysis breaks down a 6-carbon glucose molecule into two 3-carbon pyruvate
ADP, making 3-phosphoglycerate, and ATP as a byproduct. molecules, without the use of any organelles or oxygen.

So we’ll add two ATPs to our counter because this reaction happens twice - so we’re back at 0. Next, an enzyme Overall there’s a net production of 2 ATP and 2 NADH molecules, which in the mitochondria make roughly 3
called a mutase moves the phosphate on 3-phosphoglycerate to the 2nd carbon, making 2-phosphoglycerate. ATPs per NADH.

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.

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Biochemistry-metabolism
Citric acid cycle
For cells to perform any function, any work, they must have energy.
You can't go jogging or lifting weights if you’re tired, because a cell won’t work without the help of chemical
energy.
The main energy currency in the cells is adenosine triphosphate, or ATP, but any nucleoside triphosphate, like
guanosine triphosphate, GTP, will do.
For cells to make ATP, a process generating electricity has to take place in our mitochondria. Electricity is power!
And thanks to this electricity, ATP is made.
Now to create electricity, electron rich molecules must deliver electrons to a chain of complexes, the electron
transport chain, which move them to a final acceptor, a molecule of oxygen.
And there are two electron donor molecules: nicotinamide adenine dinucleotide, or NADH, and flavin adenine
dinucleotide, or FADH2.
But of course, the cell has to produce NADH and FADH2 in the first place, and they’re produced by critical
enzymes called dehydrogenases.
Dehydrogenases are the main enzymes found in the citric acid cycle or Kreb’s cycle.
In fact, the citric acid cycle is a set of 8 enzymatic reactions that start with a molecule called acetyl-CoA, and four
of the enzymes, half of them, are dehydrogenases.
And in this process, AcetylCoA gets converted into carbon dioxide.
Acetyl-CoA comes from various sources depending on whether you’ve just eaten or are starving.
Let’s say that you’re hungry and a bit angry - so you’re feeling hangry.
That’s when stress hormones like glucagon, epinephrine, and cortisol start to rise.
In this hangry state, fatty acids from triglycerides become the primary source of acetyl-CoA.
Now, let’s say you have a bowl of delicious French onion soup, everything changes - insulin is plentiful and you
have plenty of acetyl-CoA from breaking down glucose, fructose, and galactose -with glucose playing the biggest
role.
Now, alcohol is also a source of Acetyl-CoA in the liver where it’s metabolized.
In addition, proteins can also help contribute to acetyl-CoA production.
But in the case of glucose, after a meal, one glucose, a 6-carbon molecule, splits into two 3 carbon pyruvate
molecules through glycolysis, which occurs in the cytoplasm of the cell.
Each of the pyruvate molecules then enter the mitochondria.
In the mitochondria, an enzyme called pyruvate dehydrogenase snatches an electron and a carbon and two
oxygens, from pyruvate, and adds coenzyme A, making acetyl-CoA.
The electron goes to a nearby NAD+, making NADH, while the carbon and two oxygens are released to form
carbon dioxide or CO2.
This step links glycolysis to the citric acid cycle, but really isn’t considered part of either process.
Yet, it is a source of NADH and CO2 and shares some similarity with enzymes of the citric acid cycle.
As we go through the citric acid cycle, we’ll keep track of our total GTP, NADH, FADH2, and CO2 count with these
energy counters.
And remember that this cycle has many dehydrogenases.
Okay, citric acid cycle starts when acetyl-CoA is joined to a 4-carbon molecule called oxaloacetate by an enzyme
called citrate synthase, making a 6-carbon molecule - citrate. This process also releases coenzyme A.
Next, another enzyme, aconitase, rearranges the chemical shape of citrate to make its isomer, isocitrate,
without adding or removing any carbon molecules.
So far we haven’t made anything related to energy.
But here comes the first dehydrogenase, called isocitrate dehydrogenase, which removes an electron and a
carbon and two oxygens from isocitrate.
The electron goes to a nearby NAD+, making our first NADH, and the carbon and oxygens give us our first CO2,
leaving us with a 5-carbon molecule called alpha ketoglutarate.
High levels of ATP and NADH in the cell can inhibit isocitrate dehydrogenase, signaling the cycle to slow down
since the cell has plenty of energy.
On the other hand, high levels of ADP, an ATP precursor, stimulate this enzyme, signaling the cycle to speed up
since the cell needs more energy.
In fact, isocitrate dehydrogenase is considered the rate-limiting step of the cycle!
And you remember that jogging we wanted to do? Well, calcium levels rise during muscle contraction, and
contraction is work and requires energy.
And as it turns out - calcium also activates the enzyme isocitrate dehydrogenase.
Next, another dehydrogenase called alpha ketoglutarate dehydrogenase converts the 5-carbon alpha-
ketoglutarate to the 4-carbon succinyl-CoA, releasing our 2nd molecule of NADH and CO2 in the process.
Now, this enzyme requires 5 sidekicks called cofactors to function.
You can remember them by the first letters of mnemonic: T-rex Loves and Cares For Nachos.
“T” for thiamine, or vitamin B1.
“L” for lipoic acid.
“C” for coenzyme A also called vitamin B5 or pantothenate.
“F” for FAD+ also called vitamin B2 or riboflavin, and “N” for NAD+ also called vitamin B3 or niacin.
`So adequate intake of these vitamins is essential, because deficiencies can disrupt the citric acid cycle, and
impact overall health as a consequence.
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Biochemistry-metabolism
For example, thiamine deficiency can lead to a disease called beriberi, in which the central nervous system and
then the heart can’t work properly.

Likewise, niacin deficiency can cause a disease called pellagra, characterized by 4 “D”s: diarrhea, dermatitis,
dementia and, if the deficiency isn’t corrected, it can cause death. Yikes.

But let’s say that the cycle is working properly, then the next step is that an enzyme called succinate thiokinase
removes the CoA from succinyl CoA, turning it into a 4-carbon succinate molecule.

It also uses couples a phosphate and GDP molecule to the reaction, making making GTP.

Alright, next, the enzyme succinate dehydrogenase takes an electron from succinate and gives it to FAD+,
making fumarate and FADH2 in the process.

This time, we’re using FAD+ instead of NAD+ because succinate is kind of greedy and holds onto its electron
tightly.

Luckily for our cycle, FAD+ fights harder for the electron than NAD+, and is able to snag those powerful
electrons.

Also, it’s worth knowing that succinate dehydrogenase is already part of the electron transport chain, and it goes
by the name Complex II.

Next, an enzyme called fumarate hydrase or simply fumarase adds a water molecule to fumarate, making
malate.

Malate is then converted to oxaloacetate by the enzyme malate dehydrogenase, making our 3rd and final NADH
in the process.

So now we’ve come full circle, Oxaloacetate can then join up with another new acetyl-CoA molecule that’s just
hanging around waiting to begin a new cycle.

The control of the citric acid cycle is based on energy level of the cell. It has to run all the time!

So when it needs more energy, it speeds up, and when it has enough energy, it slows down.

Hormones don’t play a role in its regulation.

Summary

Alright, a quick recap. So in the end, from one acetyl-CoA molecule, we’ve made three NADH, one FADH2, one
GTP and two CO2.

The CO2s leave the cell and are transported in the blood as bicarbonate thanks to enzymes called carbonic
anhydrases.

They’re exhaled by the lungs.

In the electron transport chain, each NADH makes 3 ATPs, and each FADH2 makes 2 ATPs.

Our 1 GTP yields the energy equivalent of 1 ATP.

And so, we make a total of 12 ATP molecules per acetyl-CoA.

And since one glucose molecule splits into 2 pyruvates, each glucose molecule yields 24 ATP in the citric acid
cycle.

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Biochemistry-metabolism
Electron transport chain and oxidative phosphorylation
Your heart is constantly working. Whether you're swimming or taking a nap, your heart is always on the go.
The main form of energy that keeps our heart cells, and really all of our body cells, going is adenosine
triphosphate, or ATP.
In most cells, the main ATP producing factory is the mitochondria, which has an inner and an outer membrane,
and it’s along the inner membrane where a process called oxidative phosphorylation occurs.
“Oxidative” refers to oxidation - which is when a molecule donates its electron, and “phosphorylation” which
refers to the addition of a phosphate group to adenosine diphosphate, or ADP, to form ATP.
So oxidative phosphorylation is the process of making ATP by donating electrons to complexes embedded within
the inner mitochondrial membrane.
These complexes are proteins or lipids coupled with metals like iron and copper that facilitate the movement of
electrons.
Together, they form the electron transport chain.
During the electron transport chain, electrons are passed on from complex to complex, and finally to oxygen,
creating a proton gradient that will be used to make ATP.
The electron transport chain begins with two key molecules that want to donate their electrons: nicotinamide
adenine dinucleotide, or NADH, and flavin adenine dinucleotide, or FADH2, both of which get oxidized in the
electron transport chain.
NADH and FADH2 are primarily generated in the citric acid cycle which occurs in the mitochondria, but it can
also come directly from glycolysis - which is the breakdown of glucose in the cytoplasm, or fatty acid oxidation,
which is the breakdown of fats in the mitochondria.
Enzymes called dehydrogenases help generate the electron-rich NADH and FADH2.
And when those molecules are coming from the cytoplasm they can only enter the mitochondria using a specific
shuttle.
When using the malate-aspartate shuttle, electrons enter the electron transport chain as NADH.
When using the glycerol-3-phosphate shuttle, electrons enter electron transport chain as FADH2.
There are two entry points into the electron transport chain.
The first point of entry, is where NADH gives its electrons to Complex I.
Complex I contains flavin mononucleotide - a derivative of riboflavin or Vitamin B2 - and iron-sulfur centers
called FeS.
NADH gives its electron to flavin mononucleotide, and it gets turned back to NAD+, and can then be re-used by
dehydrogenases to make more NADH.
The second point of entry, is where FADH2 gives its electron to Complex II, which is also called succinate
dehydrogenase - the exact same enzyme that takes part in the citric acid cycle!
That means that citric acid cycle and electron transport chain actually share a step, so their activity rises and falls
together.
Ultimately, electrons from complexes I and II flow directly to Coenzyme Q, which is also called ubiquinone.
Coenzyme Q is actually a cholesterol derivative and therefore the only lipid in the electron transport chain.
Next coenzyme Q passes on the electrons to a series of cytochromes, which are proteins that contain heme
groups.
Heme groups contain iron, which is able to grab an electron and go from Fe3+ to Fe2+.
The heme iron can then release the electron to the next cytochrome in the chain and go back to Fe3+, so that
it’s ready to grab another electron.
It’s a bit like factory chain workers relaying the electrons between one another!
Complex III is made up of cytochrome b and cytochrome c1, and then electrons move over to cytochrome c, and
from there the electrons get passed to complex IV, which is composed of cytochromes a and a3, which are
together referred to as cytochrome oxidase.
Interestingly, the heme groups of complex IV contain copper rather than iron.
Cytochrome oxidase transfers the electrons to the final electron acceptor, oxygen, making the oxygen
electronegative enough to grab two protons - making a molecule of H20.
Now, if a cell doesn’t receive enough oxygen, like in hypoxia, which is the most common cellular injury, then
electron transport chain gets interrupted and ATP synthesis doesn’t happen.
So the complexes are responsible for the “oxidative” part of oxidative phosphorylation - and you can think of the
electron transport chain as a game of hot potato - with the complexes rapidly passing electrons off to one
another, creating an electrical current.
That electrical current creates energy that drives complexes I, III, and IV to pump positively charged protons out
of the mitochondria and into the space between the inner and outer mitochondrial membrane, creating a
proton gradient across the inner mitochondrial membrane.
That’s because complexes I, III, and IV are the only ones to span the inner mitochondrial membrane.
In fact, as the electrons are hopping through, the complexes are changing their conformation to push protons
across.
It’s a bit like how electrons hop wires in a home, and that energy can be used to do work - like plugging in a
vacuum to suck up dirt.
In this case, the little complexes would be the vacuums, sucking up protons.
Now, this gradient is considered unstable because the protons are always trying to equilibrate across the inner
mitochondrial membrane, even though it’s totally impermeable to them.
To get across, protons have to use a special proton channel called F0, that’s attached to an enzyme called F1. F1
is an ATP synthase that uses the proton gradient to phosphorylate an ADP molecule and make ATP - so ATP
synthase is in charge of the “phosphorylation” step of oxidative phosphorylation.
Sometimes it’s called Complex V of electron transport chain.
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Since ATP is formed in the mitochondria, it uses an ATP/ADP antiport to get pumped out of the mitochondria Another example of a medication that can be an uncoupling agent in high doses is aspirin.
and into the cytoplasm, and that way the mitochondria gets a new ADP molecule, which it uses to make the next
ATP. You can think of it as an ATP shuttle! The uncoupling can lead to a really high metabolic rate, which can lead to a metabolic acidosis, and if ATP levels
start to fall too low to allow the respiratory centers and respiratory muscles to work, then it can lead to a
Approximately 1 NADH molecule that donates its electron to the electron transport chain generates a proton respiratory acidosis as well.
gradient strong enough to make 3 ATP molecules, since NADH activates 3 proton pumps - complexes I, III and IV.
whereas, 1 FADH2 molecule only makes 2 ATP molecules, because FADH2 skips complex 1, and starts at complex A combined metabolic and respiratory acidosis quickly becomes life-threatening.
2, activating only 2 proton pumps - complexes III and IV.
Okay, the other way that oxidative phosphorylation can go awry is by inhibition, meaning that some chemicals
Since both NADH and FADH2 require an oxygen molecule to ultimately accept their electrons, these values are and drugs inhibit components of the electron transport chain.
called the phosphate to oxygen ratio or P/O ratio, which is the ratio of ATP produced per oxygen consumed for
Unlike uncoupling, inhibition stops the flow of electrons through the electron transport chain, and that leads to
each molecule. Again, these are just approximate ratios.
a decrease in ATP synthesis.
Electron transport chain isn’t controlled hormonally, rather it’s controlled by the energy level within the cell
Because electron flow is put to a stop, electron donors such as NADH and FADH2 build up, so the body doesn’t
itself.
feel the need to make more, and the metabolic rate falls.
When ATP builds up within a cell, a sign of high energy, electron transport chain slows down, and when ADP
Inhibition is like turning off the car engine, which of course means that the car won’t move.
builds up, a sign of low energy, electron transport chain speeds up.
And not surprisingly, poisons that work by inhibiting electron transport chain can lead to death pretty quickly.
Now, it turns out that drugs and chemicals can effectively “break” oxidative phosphorylation in two ways -
uncoupling and inhibition. Examples of inhibiting agents include carbon monoxide and cyanide, both of which inhibit complex IV of the
electron transport chain.
Let’s start with uncoupling. Normally, the electron transport chain is coupled with ATP synthesis, meaning that
they happen together. Barbiturates, which are GABA agonists that can be effective for seizure disorders, inhibit complex I at high doses.
Uncoupling agents break that link. Oligomycin, which is an antibiotic too toxic for human use, inhibits the F0 component of ATP synthase.
They do this by inserting their own proton channels, called ionophores, into the inner mitochondrial membrane Statins, which are a class of lipid-lowering medications don’t inhibit electron transport chain, but they can
or by simply carrying the protons back into the mitochondrial matrix, thereby allowing them to bypass the F0 decrease the synthesis of coenzyme Q.
subunit of the ATP synthase enzyme.
That reduction in coenzyme Q can result in decreased ATP production and lead to muscle pains and cramps - and
Uncoupling agents dissipate the proton gradient created by the complexes. rarely can even cause rhabdomyolysis, which is when there’s severe muscle breakdown resulting in kidney
failure from the myoglobin released.
And because the protons enter back into the mitochondria with the uncoupling agent instead of through the F0
component of ATP synthase, F1 isn’t able to phosphorylate ADP to make ATP.
Summary
Now it’s important to note that the electrons are still flowing from complex to complex all the way to the final
electron recipient, oxygen.
Alright, a quick recap. Oxidative phosphorylation is a mitochondrial process where electrons are transported
So red blood cells keep delivering oxygen to the tissues so that it can be the electron recipient. across various complexes of the electron transport chain.

Also, as the cell’s ADP levels rise, the body tries to increase its metabolic rate to make more electron donors like
NADH and FADH2.
But this electron flow is useless, because no matter how large of a proton gradient there is, the protons just go
back with the uncoupling agent instead of going through F0.
Because some of the electron’s energy is not used up in moving protons across the inner mitochondrial
membrane, more of it is available to turn into heat energy.
NADH and FADH2 are the main electron donors, and oxygen is the crucial final electron acceptor.
This movement of electrons helps establish a proton gradient that drives ATP synthase to complete the
“phosphorylation” of ADP into ATP.
Uncouplers like thermogenin disrupt oxidative phosphorylation by dissipating the proton gradient, while
inhibitors like carbon monoxide put an end to the electron transport by inhibiting electron flow.

There are some endogenous uncoupling agents like thermogenin, which is a protein found in the brown adipose
tissue of babies, used to generate heat.

Funnily enough, thermogenin is also found in hibernating animals - so yeah, we’re kind of like polar bears when
we’re babies.

Uncoupling is kind of like a car with a hot, hard working engine, but despite that, the car isn’t moving.

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Biochemistry-metabolism
Gluconeogenesis
Carbohydrates are made of sugar molecules, and the most important one is the 6-carbon sugar, glucose.
It's important to keep a steady amount of glucose in the blood, because cells use it to produce energy - in the
form of adenosine triphosphate, or ATP.
One of the ways you can do this is by eating carbohydrate-rich foods like pasta.
But in between meals, when fasting, the body maintains glucose levels using gluconeogenesis.
Gluconeogenesis is a metabolic pathway that uses enzymatic reactions to make glucose from other molecules,
like amino acids, lactate, and glycerol.
Gluconeogenesis primarily takes place in liver cells, but it can also happen in the epithelial cells of the kidney and
the intestines.
Specifically, it takes place in the cytoplasm, mitochondria, and endoplasmic reticulum of cells found in these
tissues.
Okay, so let’s say you’re going hiking in the woods, and you fuel up on some pasta before you leave. Now, during
the hike, you get lost and end up stranded with no food. Initially, the glucose in your pasta is broken down by a
series of enzymatic reactions to make, producing ATP in the process.
This is called glycolysis, and it keeps you going for a couple of hours.
Some of the extra glucose is stored in the liver cells in the form of glycogen, which is a bunch of glucose
molecules stringed together.
When you’re fasting, you still need glucose, in particular for your red blood cells and your brain. And you might
need it to find your way out of the woods.
So, it’s up to your liver to maintain adequate blood glucose levels while fasting.
There are two pathways that can contribute glucose: glycogenolysis and gluconeogenesis.
They both start at the very same time, when the liver has enough energy to do this job!
The liver gets energy exclusively from burning fat, and as soon as its ATP levels are high enough, the liver will
start releasing glucose in the blood for extrahepatic tissues to use.
The liver will break down glycogen into individual glucose molecules, in a process called glycogenolysis, but that
only helps for 12 to 24 hours of fasting, because glycogen stores are finite.
In contrast, gluconeogenesis makes glucose from scratch, so it can keep on going in the event that you fast for
more than a day.
Actually, by 12 hours of fasting, gluconeogenesis is the main provider of glucose in the bloodstream.
The process of gluconeogenesis is almost reverse glycolysis.
In glycolysis, you’re using 10 enzymatic reactions to convert glucose to pyruvate in order to make ATP.
And in gluconeogenesis, you’re working backwards and using ATP to convert pyruvate to glucose.
7 of the reactions in glycolysis and gluconeogenesis are reversible, meaning they can go in both directions using
the same enzymes.
However, the other 3 are irreversible, meaning that the enzyme mediating the reaction can only go in the
direction of glucose to pyruvate, but not the opposite.
So if you think of the path between glucose and pyruvate as a two-way road within the cell.
When driving from glucose to pyruvate, it’s a smooth straightforward ride.
On the other hand, when driving from pyruvate to glucose, most of the ride is straightforward, but there are 3
roadblocks which represent irreversible reactions.
And for those, you have to take an alternate route to bypass them, which represent unique reactions that use
different enzymes only found in gluconeogenesis.
Alright, so the ingredients we need to cook up some glucose from scratch in our liver are a source of pyruvate
and ATP.
In gluconeogenesis, the two main sources of pyruvate are lactate and amino acids like alanine.
Lactate is produced as a byproduct of anaerobic respiration in red blood cells and exercising skeletal muscle
cells.
An enzyme called lactate dehydrogenase removes a hydrogen from lactate, turning it into pyruvate.
In that reaction, the hydrogen goes to a nearby nicotinamide adenine dinucleotide, or NAD+, which turns into
NADH.
The second source of pyruvate is amino acids, which are the building blocks of proteins.
We have 20 different amino acids, and 18 of them, with the exception of leucine and lysine, are glucogenic
amino acids, meaning they can be used to make glucose.
You can remember the exceptions - leucine and lysine - as the “Lazy Ls” of amino acids.
When fasting for a long time, the body breaks down protein in skeletal muscle cells into individual amino acids,
with the main amino acid being alanine.
The enzyme alanine transaminase, or ALT, removes an amino group from alanine, and turns it into pyruvate.
The amino group attaches to an acid called alpha ketoglutarate, which then turns into glutamate.
And in general, transaminase enzymes require pyridoxine, or vitamin B6 as a cofactor.
Now, to get ATP when you’re stranded in the woods, your body starts breaking down fats which come in the
form of triacylglycerides.
During fasting, the pancreatic alpha cells sense blood glucose levels decreasing, and release glucagon.
In the meantime, the low level of insulin, the presence of epinephrine, as well as ACTH are causing adipocytes or
fat cells to stimulate hormone-sensitive lipase or HSL, an enzyme that breaks triacylglycerides down into three
fatty acids and glycerol.
The fatty acids enter the bloodstream and go to the hepatocyte mitochondria, where they’re broken down into
acetyl CoA and ATP by a metabolic process called beta oxidation.
The glycerol, on the other hand, is used to make glucose in gluconeogenesis.
So always remember, lactate, glycerol and all amino acids except leucine and lysine will be used to build glucose.
Now let’s cook up some glucose!
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The first step is converting pyruvate to phosphoenolpyruvate, or PEP - but this is also our first roadblock.
In glycolysis, the enzyme pyruvate kinase catalyzes PEP turning into pyruvate, but not vice versa.
The alternate route involves 3 enzymes; pyruvate carboxylase, malate dehydrogenase, and
phosphoenolpyruvate carboxykinase, or PEPCK.
So the alternate route starts when pyruvate enters the mitochondria, and pyruvate carboxylase adds a carbon to
it, making a 4-carbon molecule, oxaloacetate.
A general rule of thumb is that any carboxylase enzyme requires 3 cofactors, which you can remember with an
ABC mnemonic, “a” for ATP, which comes from fatty acid oxidation, “b” for biotin, or Vitamin B7, and “c” for
carbon dioxide.
Also, the acetyl-CoA that’s made during fatty acid oxidation enhances pyruvate carboxylase activity.
So now we have oxaloacetate, and it needs to get out of the mitochondria and into the cytoplasm to continue
gluconeogenesis.
Unfortunately, oxaloacetate can’t go through the mitochondrial membrane - so malate dehydrogenase in the
mitochondria comes to the rescue and adds a hydrogen to oxaloacetate, converting it to malate.
Malate can exit the mitochondria and enter the cytoplasm, where malate dehydrogenase in the cytoplasm
converts it back to oxaloacetate.
This process of conversion and reconversion is called the malate shuttle.
Okay, now that oxaloacetate is in the cytoplasm, PEPCK removes a carbon group, and adds a phosphate group,
turning oxaloacetate into PEP.
This reaction requires an energy source, but instead of ATP, the energy comes in the form of guanosine
triphosphate, or GTP.
Don’t worry, it doesn’t make a difference since the energy is contained in the phosphate group.
So-called “stress” hormones, like glucagon, epinephrine, and cortisol enhance the activity of PEPCK by induction-
and being stranded in the woods generates a lot of these hormones, speeding up gluconeogenesis.
Now that we have PEP, it goes through a series of reversible reactions, which are common to glycolysis, until it’s
converted to dihydroxyacetone phosphate - or DHAP.
Alternatively, glycerol from hormone-sensitive lipase activity can also be used to make DHAP.
In that process, the enzyme glycerol kinase adds a phosphate group from ATP to the 3rd carbon of glycerol,
making glycerol-3-phosphate, or G3P.
Then, another enzyme called glycerol-3-phosphate dehydrogenase converts G3P to DHAP.
Either way, once DHAP is formed, it’s converted to fructose 1,6 bisphosphate by a reversible reaction involving
the enzyme aldolase.
Now, here’s our second roadblock. In glycolysis, the enzyme phosphofructokinase-1, or PFK1 adds a phosphate
group to fructose-6-phosphate, making fructose-1,6-bisphosphate.
In gluconeogenesis, there’s a different enzyme called fructose-1,6-bisphosphatase, that removes a phosphate
from the 1st carbon of fructose-1,6-bisphosphate, making fructose-6-phosphate.
This reaction is the rate limiting step of gluconeogenesis, meaning that how fast fructose 1,6 bisphosphatase
catalyzes this reaction determines the speed at which all of gluconeogenesis happens.
As a result, this step is really tightly controlled to make sure we make glucose when we need to, and stop when
we don’t.
For example, one trigger that speeds up gluconeogenesis is when the liver is burning up a lot of fatty acids,
yielding a lot of ATP.
This ATP enhances the activity fructose-1,6 bisphosphatase.
Remember, the liver needs energy to carry out gluconeogenesis!
It also relies on the presence of stress hormones to enhance the activity of PEPCK.
Glucagon activates whereas insulin inhibits fructose-1,6 bisphosphatase.
So next, fructose-6-phosphate is converted to its isomer, glucose-6-phosphate by the enzyme isomerase, which
changes the molecular shape, but not its chemical structure. That’s why the phosphate on the 6th carbon is still
there.
But glucose can’t go into the bloodstream with a phosphate clinging to it, so cutting it off is the final roadblock.
In glycolysis, when glucose enters a cell, the enzyme hexokinase adds a phosphate to glucose to keep glucose
inside the cell.
Removing the phosphate off of the 6th carbon of glucose, requires an enzyme located in the endoplasmic
reticulum called glucose-6-phosphatase.
Once the phosphate is removed, glucose can enter the bloodstream.
Now - one final thought on gluconeogenesis relates to chronic alcoholism.
Alcohol, or ethanol is also processed in liver cells, and over time it results in an accumulation of NADH and ATP in
the cytoplasm.
Increased NADH levels signal the lactic, malate, and glycerol-3-phosphate dehydrogenase enzymes in
gluconeogenesis to go the other way around: from pyruvate to lactate, from oxaloacetate to malate and from
DHAP to G3P, respectively.
On the other hand, high ATP levels in the liver slow down fatty acid beta oxidation and cause abnormal
accumulation of fat in the liver.
Both of these effects impair gluconeogenesis, resulting in chronic low blood sugar when fasting - not a good way
to get stranded in the woods.
Summary Alright, a quick recap. Gluconeogenesis involves the production of glucose from pyruvate
to help maintain steady blood glucose levels during prolonged fasts.
In essence, it is reverse glycolysis, but there are 3 irreversible reactions that are bypassed using enzymes that are
unique to gluconeogenesis.
Pyruvate kinase is bypassed by pyruvate carboxylase, malate dehydrogenase and PEPCK.
PFK1 is bypassed by fructose-1,6-bisphosphatase, and hexokinase is bypassed by glucose-6-phosphatase.
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Biochemistry-metabolism
Glycogen metabolism
Glucose is a 6-carbon molecule that's used to make energy, in the form of adenosine triphosphate, or ATP.
Glucose is such an important energy source, that our body stores excess glucose in skeletal muscle cells and liver
cells in the form of glycogen.
Glycogen is basically an enormous molecule or polymer, that’s made up of glucose molecules linked together by
glycosidic bonds.
You can think of glycogen having a main chain, and there being multiple branches sprouting off of it.
These branches allow glycogen to be compact and capable of rapid addition and removal of glucose.
It’s a bit like growing a plum tree in a tiny house with a short ceiling.
The short ceiling limits the tree’s vertical growth, but the tree’s able to branch off, so that it can still grow and
produce many plums in a tight space.
Now let’s say that you just wrapped up a delicious lunch - you had tacos! Glucose is absorbed from the intestine
and our blood sugar goes up. The pancreas responds to high blood sugar by secreting insulin.
Insulin acts on glucose transporters on the cell membrane, which are called GLUTs - and makes them bring more
glucose into all the cells in our body.
Inside the cell, an enzyme called hexokinase adds a phosphate group to it’s 6th carbon, creating glucose 6
phosphate.
Then, glucose-6-phosphate is broken down during glycolysis, making ATP as a byproduct.
Over time, ATP levels start to rise and that inhibits certain enzymes in glycolysis.
When that happens, the extra glucose-6 phosphate can be used to make glycogen. And that usually takes place
in the liver and muscle cells.
There are four main steps in glycogen synthesis.
First is attaching a uridine diphosphate, or UDP molecule to glucose.
Second, is attaching the glucose part of the UDP-glucose molecule to a glycogen primer called glycogenin,
forming a short linear glycogen chain, which serves as a primer.
Third, is adding more glucose molecules to that primer - a bit like forming a conga line.
And fourth, is adding branches to the glycogen molecule.
So starting with step one, to make UDP-glucose, an enzyme called phosphoglucomutase moves the phosphate
from the 6th carbon of glucose-6-phosphate to the 1st carbon, creating glucose-1-phosphate.
Next, we’ll need energy - which, uniquely, comes in the form of uridine triphosphate, or UTP.
In the presence of glucose-1-phosphate and UTP, an enzyme called UDP-glucose pyrophosphorylase cuts two
phosphate molecules off of UTP, which give the energy necessary to complete this reaction.
So only one phosphate remains attached to uridine, and then glucose-1-phosphate is added to it.
That makes two phosphates. So the resulting molecule is called UDP-glucose.
Once many glucose molecules are converted into UDP-glucose molecules, we’re ready to create glycogen.
An enzyme called glycogen synthase catalyzes the attachment of the glucose part of UDP-glucose to another
glucose residue at the end of glycogen branch, forming an alpha 1,4 glycosidic bond. It’s almost as if the glucose
molecules are holding hands!
And in addition to prolonging the glycogen chain, there’s another byproduct of this reaction is UDP.
But, it turns out that glycogen synthase can only elongate an already existing glycogen chain that’s at least 4
glucose molecules long.
So, if there aren’t at least four glucose molecules linked up together already, then glycogen synthesis requires a
protein called glycogenin.
Glycogenin plays the role of fooling glycogen synthase by catalyzing the attachment of 4 glucoses to itself,
creating a short chain connected with alpha 1,4 glycosidic bonds.
By doing that, it’s able to tell glycogen synthase “Hey, we have a chain here that kind of looks like an old
glycogen molecule”.
Glycogen synthase falls for it, and elongates this short chain on glycogenin by attaching lots of glucose molecules
to it through alpha 1,4 glycosidic bonds. This elongates the chain and creates a new linear glycogen molecule.
Next, there’s an enzyme called the branching enzyme, goes to the ends of the chain and cuts off a chain of about
6 to 8 glucose residues in length.
The branching enzyme then attaches that chain to the side of the linear glycogen strand by creating an alpha 1,6
glycosidic bond - so there’s now a bond between the 1st carbon of the glucose on the small cleaved segment
and the 6th carbon of a glucose that’s part of the linear chain.
And as soon as you’ve shortened the linear chain, glycogen synthase will elongate it once again.
This happens over and over again, resulting in a branched glycogen tree to serve as stored energy.
Now let’s say it’s been a couple of hours since those tacos, and you decide to go for a run.
Because you’re fasting, your blood glucose levels take a dip.
In response, the pancreas secretes the hormone glucagon and the adrenal glands secrete epinephrine to
increase your heart rate.
It turns out that glucagon tells the liver cells to break glycogen down into individual glucose molecules, and
epinephrine tells skeletal muscle cells to do the same.
In both the liver and skeletal muscle cells, glycogen breakdown begins with the branches.
First, an enzyme called glycogen phosphorylase cleaves the alpha 1,4 bonds between individual glucose residues
and catalyzes the transfer of a phosphate group to the freed glucose.
The result is that the enzyme releases one glucose-1-phosphate molecule at a time.
It keeps on doing this until exactly 4 glucose molecules remain on the branch.
Next, a debranching enzyme literally cuts off glycogen branches.
It has a component called 4-alpha-glucanotransferase, which transfers 3 out of the 4 glucose molecules off of
the branch and reattaches them to the linear glycogen chain instead, extending it as a result.
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The same debranching enzyme has another component known as alpha 1,6 glucosidase, which cleaves off the
alpha 1, 6 glycosidic bond and releases a free glucose.

So for each glucose that’s removed via phosphorolysis, there’s a glucose-1-phosphate that gets liberated, and
it’s converted to glucose-6-phosphate by phosphoglucomutase.

The difference between glycogen breakdown in the liver and what goes on in the muscles results from the
different enzymes in those two tissues.

In liver cells, glucose-6-phosphatase removes the phosphate off of the 6th carbon, releasing free glucose into
the bloodstream, for other organs and tissues to use.

Skeletal muscles don’t have this enzyme, so they simply use the glucose-6-phosphate by sending it into the
glycolysis pathway to make energy that can help you with that run.

Glycogen metabolism is primarily regulated the two pancreatic hormones - insulin and glucagon.

Now a general rule of thumb is, glycogen synthase is active when it doesn’t have a phosphate, whereas glycogen
phosphorylase is active when it does have a phosphate attached to it.

So in liver and skeletal muscle cells, insulin binds to a tyrosine kinase receptor on the cell surface, and that
ultimately activates a protein phosphatase which goes around removing phosphates from glycogen synthase,
making it active, and from glycogen phosphorylase, making it inactive.

This promotes glycogen synthesis and decreases its’ breakdown.

On the other hand, glucagon in the liver cells binds to a G-protein coupled receptor on the cell surface, which
activates adenylyl cyclase, which converts ATP to cyclic AMP, or cAMP.

cAMP then activates protein kinase A which adds a phosphate to glycogen phosphorylase kinase, activating it.

Glycogen phosphorylase kinase adds a phosphate to glycogen phosphorylase increasing its’ activity and
promoting glycogen breakdown.

It also adds a phosphate to glycogen synthase, decreasing its activity and therefore decreasing glycogen
synthesis.

Summary

Alright, a quick recap. Glycogen is a multi branched, compact structure that’s made of alpha 1,4 glycosidic bonds
between the glucose molecules, and alpha 1,6 bonds at the branching points.

Glycogen is considered the major form of glucose storage in the body.

It’s primarily stored in the liver cells and skeletal muscle cells.

After a meal, high insulin levels promote glycogen synthesis, whereas during fasting, high glucagon and
epinephrine levels promote glycogen break down.

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Biochemistry-metabolism
Pentose phosphate pathway
Let's say you just ate a carbohydrate loaded meal, like a bowl of rice.
A few hours after you’re done, those carbohydrates are broken down in the small intestine into their simplest
chemical form; monosaccharides, the most important of which is glucose - a 6-carbon molecule that’s in the
shape of a ring.
Glucose moves from the small intestine into the bloodstream, and blood glucose levels rise, which causes the
pancreas to secretes a hormone called insulin.
Insulin makes more glucose enter cells through specific transporters called GLUTs.
Once glucose is in the cell, an enzyme called hexokinase attaches a phosphate group to its sixth carbon, creating
glucose-6-phosphate.
From there, the cell has the option to take glucose through a metabolic pathway called glycolysis; which is the
breakdown of glucose in order to generate ATP.
But if the cell doesn’t need ATP, glucose can be used to make some other useful products by entering an
alternative metabolic pathway called the pentose phosphate pathway.
The pentose phosphate pathway is named for the products it ultimately generates; pentose refers to a five-
carbon sugar called ribose, and phosphate refers to a molecule called nicotinamide adenine dinucleotide
phosphate, or NADPH.
So the pentose phosphate pathway is an alternative pathway that glucose can enter when cells need to make
more ribose and NADPH.
Ribose can be used to make nucleotides, which are the building blocks of our DNA and RNA.
And NADPH is rich in electrons, and can be used in various anabolic pathways.
Anabolic pathways are ones that synthesize molecules like fatty acids, from scratch, and require an electron
donor - such as NADPH.
Like glycolysis, the pentose phosphate pathway happens exclusively in the cytoplasm and it doesn’t require any
special organelles which means that all of our cells can use this pathway.
The pentose phosphate pathway can be divided into two phases: an irreversible oxidative phase that ultimately
yields NADPH, and a reversible non-oxidative phase that yields ribose.
Irreversible means that the reaction can only go in one direction - that is, substrate to product.
On the other hand, reversible means that the reaction can go in both directions, and the substrate and product
can be interconverted into one another, depending on what the body needs more.
Oxidation of a molecule means that a molecule donates or loses its electrons - in the form of hydrogens - to
another molecule.
Okay, to launch the oxidative phase, an enzyme called glucose-6-phosphate dehydrogenase, or G6PD, snatches a
hydrogen from glucose-6-phosphate, and offers it to NADP+, making 6-phosphogluconate and NADPH in the
process.
This is the rate-limiting step of the pentose phosphate pathway.
The rate-limiting step of any reaction is the step that takes the longest to happen, so it determines the overall
rate of the pathway.
The next step involves an enzyme called 6-phosphogluconate dehydrogenase, which, much like G6PD, steals an
electron from 6-phosphogluconate and offers it to another NADP+, making our second NADPH.
But unlike G6PD, this enzyme also removes a carbon from the 6-carbon 6-phosphogluconate and releases it as
carbon dioxide, making a 5-carbon sugar called ribulose-5-phosphate.
This marks the end of the oxidative phase, with a total of 2 NADPH molecules created per glucose.
Now, since NADPH is electron-rich, it’s time for it to give back to the cellular community.
NADPH can donate electrons in anabolic pathways such as fatty acid synthesis, steroid hormone synthesis, and
cholesterol synthesis.
So there’s usually a lot of NADPH in tissues that make these molecules, like the liver, the adrenal cortex, the
gonads, and the milk-producing breast glands.
In addition to anabolic pathways, NADPH is also a key player for antioxidant reactions.
For example, red blood cells normally produce reactive oxygen species such as hydrogen peroxide as a
byproduct of oxygen metabolism.
Hydrogen peroxide can be harmful, so red blood cells also produce antioxidants such as glutathione, which can
neutralize hydrogen peroxide.
In the presence of an enzyme called glutathione peroxidase, glutathione donates electrons to hydrogen
peroxide, converting it to water, which is harmless.
NADPH comes into play as an electron supplier.
In the presence of an enzyme called glutathione reductase, NADPH donates its electrons to glutathione, so that
it may effectively neutralize hydrogen peroxide.
So if NADPH wasn’t generated by the pentose phosphate pathway, glutathione wouldn’t get the electrons it
needs, and hydrogen peroxide would accumulate and cause damage to proteins within the cell, eventually
leading to cell death.
Now, let’s go back and see what happens to ribulose-5-phosphate during the non-oxidative pathway.
Now, that’s ribulose, not ribose.
The cool aspect about the non-oxidative pathway is that is quite flexible and reversible.
It can generate different products depending on what the cell needs.
Ribulose-5-phosphate initially gets converted to ribose-5-phosphate by an isomerase enzyme.
An isomerase simply shuffles the carbons on the molecule into different positions without adding or removing
anything, similar to shuffling a deck of cards.
Ribose-5-phosphate is important in nucleotide synthesis, which are the building blocks of DNA and RNA.
Nucleotides are made up of a 5-carbon sugar, a phosphate backbone and a purine or pyrimidine nitrogen base.
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So ribose-5-phosphate serves as that 5-carbon sugar required for DNA or RNA synthesis.

Alternatively, ribulose-5-phosphate can also be converted into another 5-carbon sugar, called xylulose-5-
phosphate, by an enzyme called an epimerase.

Now let’s say the cell doesn’t need to make DNA or RNA - here’s where the non-oxidative phase gets really
playful, with the help of two other enzymes: transketolase and transaldolase.

Transketolase removes 2 carbons from xylulose-5-phosphate, and requires vitamin B1, or thiamine as a cofactor.

The 2-carbons go to ribose-5-phosphate, creating a 7-carbon molecule called sedoheptulose-7-phosphate, as

well as a 3-carbon molecule called glyceraldehyde-3-phosphate.

Now transaldolase comes in and removes 3-carbons from sedoheptulose-7-phosphate, turning it into a 4-carbon
molecule called erythrose-4-phosphate.

The 3-carbons go to glyceraldehyde-3-phosphate, making the 6-carbon fructose-6-phosphate.

If there’s another xylulose-5-phosphate around, transketolase can jump in and remove 2 carbons from it, giving
them to erythrose-4-phosphate to make another fructose-6-phosphate molecule.

So processing 3 ribulose 5-phosphate molecules can yield one glyceraldehyde-3-phosphate and two fructose-6-
phosphates.

Both glyceraldehyde-3-phosphate and fructose-6-phosphate are intermediate molecules in glycolysis.

So, even in the middle of the pentose phosphate pathway, the cell can decide to divert towards glycolysis if it
needs more energy, and vice versa; if the cell is in the middle of glycolysis, but it realizes it needs to craft some
nucleotides instead, transketolase and transaldolase can convert G3P and fructose 6 phosphate back to ribulose-
5-phosphate.

Summary

Alright, a quick recap. The pentose phosphate pathway is an alternative metabolic pathway for glucose.

It’s made up of an oxidative phase, which makes NADPH, and a non-oxidative phase, which makes ribose-5-
phosphate.

NADPH is an important electron donor, and is required by anabolic reactions as well as antioxidant molecules.

Ribose-5-phosphate is crucial for making the nucleotides necessary for DNA and RNA synthesis, as well as
creating intermediates of glycolysis, like G3P and fructose-6-phosphate.

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Fatty acid oxidation
Our bodies are capable of surviving without food for long periods of time, up to 2 weeks!
The reason we can do that is that we can store our dietary fuels, and then break them down when needed to
make energy in the form of adenosine triphosphate, or ATP.
Fat is one of the most important ways we store energy and the term “burning fat”, actually refers to fatty acid
oxidation.
In fact, if two individuals were stranded in the Andes mountains with no food, the person with more fat content
would survive longer - yet another reason to avoid working out.
What makes fat such a great source of energy are fatty acids, which are the simplest form of fats, composed of
long chains of carbon and hydrogens.
The transfer of electrons in the form of hydrogen molecules from these fatty acids to certain molecules, can
then be used to generate ATP.
Fatty acid oxidation primarily takes place in the mitochondria of heart, skeletal muscle, and liver cells.
Before we can oxidize fat, it needs to be moved from storage sites to the cells that can use it.
Fat is stored in adipocytes or fat cells as triglycerides, which are 3 fatty acids attached to a glycerol molecule.
Triglycerides can be broken down by the enzyme hormone sensitive lipase, into free fatty acids and glycerol.
So if you're starving in the Andes, first your blood glucose level falls.
In response, the pancreas secretes a hormone called glucagon which increases the activity of hormone sensitive
lipase, and increases the breakdown of triglycerides.
Now, the free fatty acids can leave the fat cell, and enter the bloodstream, where they bind to a protein called
albumin.
Albumin carries the fatty acids to target cells, like liver cells, that are capable of fatty acid oxidation.
First, the free fatty acid dissociates from albumin and diffuses into the cell.
Once inside the cell, a cytosolic enzyme called fatty acyl-CoA synthetase adds a coenzyme A molecule to the end
of the fatty acid, turning it into a metabolically active fatty acyl-CoA.
This process requires 2 ATP molecules - so it takes a little energy to make energy, but it’s totally worth it in the
end.
Now the mitochondria is composed of two membranes - an outer membrane and an inner membrane, with a
small space in between - and the mitochondrial matrix at the core.
The enzymes required for beta oxidation are located in the mitochondrial matrix - however, the fatty acid can’t
cross the inner mitochondrial membrane when CoA is attached to it.
To get around this problem, we need to free the CoA from the fatty acid.
An enzyme within the outer mitochondrial membrane called carnitine acyltransferase 1, or CAT1, replaces the
CoA with a carnitine, making fatty acyl-carnitine and a free CoA, both of which can easily cross the inner
mitochondrial membrane.
Then, along the inner mitochondrial membrane, another enzyme called carnitine acyltransferase 2, or CAT2,
substitutes carnitine and CoA back, therefore regenerating fatty acyl-CoA and free carnitine - which is now
within the mitochondrial matrix.
This whole slick process is called the carnitine shuttle.
Carnitine can cross the inner mitochondrial membrane by itself, so it can go back to the outer membrane to
meet the next incoming fatty acid.
We can also get more carnitine from our diet, mainly from meat products.
Finally, the carnitine shuttle can be regulated by a product of fatty acid synthesis called malonyl-CoA, which
specifically inhibits CAT1, slowing down fatty acid oxidation.
After all, you don’t want to make and break fatty acids at the same time.
Okay, so fatty acid chains can vary in length, from short, medium, long and very long fatty acids, but the process
of fatty acid oxidation is the same for any length.
We’ll explain fatty acid oxidation with an example of a long chain fatty acid: the 16-carbon long palmitoyl-CoA.
The enzymatic modifications that take place in fatty acid oxidation happen on the 2nd and 3rd carbon of the
chain, also called the alpha and beta carbons, respectively.
Each of those carbons enters fatty acid oxidation with 2 hydrogens bound to it.
First, an enzyme called acyl-CoA dehydrogenase removes 1 hydrogen from the 2nd carbon, and one hydrogen
from the 3rd.
The same enzyme then gives those 2 hydrogens to a nearby flavin adenine dinucleotide molecule, or FAD,
making FADH2, and converting the palmitoyl-CoA to enoyl-CoA in the process.
Enoyl-CoA has only 1 hydrogen on each of the alpha and beta carbons.
This is the first oxidation step.
Next, an enzyme called enoyl-CoA hydratase transfers a hydroxyl group, which is an oxygen linked with a
hydrogen, from a water molecule on to the beta carbon of enoyl CoA, making beta-hydroxyacyl-CoA.
The second oxidation step involves a similar hydrogen transaction mediated by another dehydrogenase called
beta-hydroxyacyl-dehydrogenase.
Beta-hydroxyacyl-dehydrogenase removes two hydrogens from the beta carbon, and transfers one of them to a
nicotinamide adenine dinucleotide molecule, or NAD+, making NADH and beta-ketoacyl-CoA in the process, and
releasing the other hydrogen.
And because this oxidation step occurs at the beta-carbon, fatty acid oxidation is also sometimes called beta-
oxidation.
Finally, an enzyme called beta-ketothiolase cleaves those 2 alpha and beta carbons off the fatty acid chain by,
making a 2-carbon acetyl-CoA molecules in the process.
This leaves us with a 14-carbon fatty acid chain that can enter another cycle of beta-oxidation.
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So the longer the fatty acid chain, the more cycles, and the more energy we make. Alright, a quick recap. Fatty acids are one of the main sources of energy in the body, especially during the fasting
state.
And in one cycle of beta-oxidation, we made 1 NADH, 1 FADH2 and 1 acetyl-CoA.
Fatty acids oxidation occurs in 3 steps: activation, then transport through the carnitine shuttle, and finally beta-
NADH and FADH2 are now full of electrons, and can enter a mitochondrial pathway called the electron transport oxidation in the mitochondrial matrix.
chain, where those electrons can be used to make ATP.

1 NADH molecule makes roughly 3 ATPs, while 1 FADH2 molecule makes roughly 2 ATPs.

On the other hand, acetyl-CoA can enter the citric acid cycle - which is another metabolic pathway in the
mitochondria that burns acetyl-coA to make more NADH and FADH2, which then enter the electron transport
chain, yielding a total of 12 ATPs per acetyl-CoA.

So from a 16-carbon palmitoyl-CoA molecule, we make a total of 7 NADH, 7 FADH2 and 8 acetyl-CoA molecules.

If we calculate the total ATP yield, we make 131 ATPs.

But remember we consumed 2 ATPs when we first activated fatty-acyl CoA.

So per palmitoyl-CoA, we made a net worth of 129 ATP molecules. That’s a lot of ATP. Told you it was worth it.

Now that’s straightforward enough for a fatty acid chain that has an even number of carbons, but the oxidation
of a fatty acid with an odd number of carbons can be somewhat different.

So let’s say we’re dealing a 13-carbon fatty acid.

Initially, the process of oxidation goes exactly the same, until we reach a 3-carbon fragment called propionyl-
CoA.

This is when it gets a bit awkward for the prototypical oxidation enzymes, so to get the job done, we’ll need 3
other enzymes to break it down. 3 reactions for the 3-carbon molecule.

First, an enzyme called propionyl-CoA carboxylase adds a carboxyl group to propionyl-CoA, making
methylmalonyl-CoA.

This enzyme requires 3 cofactors, which can be remembered with a simple “ABC” mnemonic.

“A” for ATP, “B” for biotin, or vitamin B7 and “C” for carbon dioxide, which is the carboxyl group source in this
reaction.

Next, an enzyme called methylmalonyl-CoA mutase rearranges the carbons on methylmalonyl-CoA to make
succinyl-CoA.

This enzyme requires vitamin B12 as a cofactor.

Succinyl-CoA can then enter the citric acid cycle, or it be used for the synthesis of heme, which is the oxygen
binding element in hemoglobin.

Finally, for really long fatty acid molecules that are 22-carbons long or longer, specific organelles called
peroxisomes may be needed.

Peroxisomal oxidation works the same way as mitochondrial oxidation, but just has different enzymes that are
capable of degrading the long fatty acid chain until it’s under 22 carbons - and then the fatty acids are sent back
to the mitochondria to take over.

Summary

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Fatty acid synthesis
In addition to carbohydrates and proteins, lipids are the third main macromolecule we consume in our diet.
Fatty foods include red meat, dairy products, and even peanut butter.
And lipids come in many forms, including cholesterol, glycerol, phospholipids, and fatty acids.
Of these, fatty acids are the simplest form of lipids - they're basically just long chains of carbon and hydrogen,
that are grouped by length into short, medium, long and very long chain fatty acids.
Fatty acids can also combine with glycerol to make triacylglycerides, which is made of 3 fatty acids attached to a
glycerol molecule, and is the main storage form of fat in our body.
Now, short and medium-chain fatty acids are primarily obtained from the diet, but the liver and fat cells can
synthesize long chain fatty acids.
This occurs by combining lots of 2-carbon molecules, called acetyl-coenzyme A or acetyl-CoA, into a single 16-
carbon, long chain fatty acid called palmitoyl-coenzyme A, or palmitoyl-CoA.
Palmitoyl-CoA can then serve as a precursor to even longer chain fatty acids.
To make palmitoyl-CoA, acetyl-CoA provides the carbon atoms, and nicotinamide adenine dinucleotide
phosphate, or NADPH provides the hydrogen atoms.
As it turns out, most of the acetyl-CoA used to make fatty acids comes from carbohydrate metabolism -
specifically glucose, which is a 6-carbon sugar molecule.
After eating a glucose-rich dinner, like cake and cookies, glucose levels in the blood rise quickly.
In response, the pancreas secretes insulin, a hormone which makes our cells take in and process a lot more
glucose.
Inside the cells, glucose can enter glycolysis where it’s broken down into two 3-carbon pyruvate molecules, and
that yields a bit of energy in the form of adenosine triphosphate - or ATP.
Pyruvate then moves into the mitochondria, and is converted to acetyl-CoA by an enzyme called pyruvate
dehydrogenase.
Inside the mitochondria, acetyl CoA enters the citric acid cycle by combining with a molecule called
oxaloacetate, to form citrate.
Citrate can then continue in the citric acid cycle, which generates electron carriers that can join the electron
transport chain and oxidative phosphorylation.
All of this leads to the formation of a lot more ATP.
So the math is simple - more glucose, more ATP.
Well, ATP inhibits some enzymes in the citric acid cycle, slowing it down overall, and that means that extra
acetyl-CoA can be used to make fatty acids instead.
However, the enzymes required for fatty acid synthesis are all in the cytoplasm, so in order to start fatty acid
synthesis, acetyl-CoA needs to get out of the mitochondria.
Unfortunately, acetyl-CoA cannot cross the mitochondrial membrane - so to get to the cytoplasm, it combines
with oxaloacetate to form citrate, just as it would to enter the citric acid cycle.
So when there’s a lot of ATP around, citrate crosses the mitochondrial membrane and enters the cytoplasm.
In the cytoplasm, an enzyme called citrate lyase, cleaves citrate back into acetyl-CoA and oxaloacetate.
This process of conversion and reconversion is called the citrate shuttle.
In the meantime, oxaloacetate is recycled and goes back into the mitochondria so it can be available the next
incoming acetyl-CoA.
But we have another problem; oxaloacetate can’t cross the membrane either.
So an enzyme called malic enzyme, converts oxaloacetate into pyruvate, forming NADPH from NADP+ in the
process.
Now, pyruvate can cross the mitochondrial membrane, and an enzyme called pyruvate carboxylase converts it
back into oxaloacetate, which can begin a new cycle.
High levels of acetyl-CoA also increase the activity of pyruvate carboxylase, so that oxaloacetate is made
available.
With acetyl-CoA in the cytoplasm, all we need to begin fatty acid synthesis is NADPH to provide the hydrogens.
Some of that NADPH is generated by malic enzyme when it converts oxaloacetate to pyruvate.
And the rest comes from the metabolism of the excess glucose in a pathway called the pentose-phosphate
pathway.
Once there’s enough NADPH, acetyl-CoA can begin its journey towards palmitoyl-CoA.
Ok, so first, a carboxyl group is added to acetyl-CoA by an enzyme called acetyl-CoA carboxylase, converting it to
the 3-carbon malonyl-CoA.
This enzyme requires 3 cofactors, which can be easily remembered with the mnemonic, ABC.
“A” is for ATP, “B” is for biotin, or vitamin B7, and “C” is for carbon dioxide, or CO2, which is the carboxyl group
source.
However, this additional carbon will not contribute to the fatty acid chain, because it’s lost later on.
This is considered the rate-limiting step of fatty acid synthesis.
Which means that the speed of this reaction will determine the overall rate at which all of fatty acid synthesis
happens - so acetyl CoA carboxylase is tightly regulated.
There are two types of regulation - hormonal regulation and allosteric regulation.
Hormonal regulation involves the pancreatic hormones insulin and glucagon - and they work by adding or
removing a phosphate group on acetyl CoA carboxylase.
When insulin is released, like after that cake and cookie bonanza, it activates the enzyme protein phosphatase 2,
which removes a phosphate group from acetyl-CoA carboxylase increasing its activity.
On the other hand, when glucagon is released like when you’re fasting, it activates an enzyme called adenosine
monophosphate, or AMP-dependent kinase, which adds a phosphate group to acetyl-CoA carboxylase
decreasing its activity.
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So glucagon basically “handcuffs” the enzyme, and insulin sets it free. But in the first step, we placed an additional acetate molecule on the ACP group, instead of converting it to
malonyl-CoA.
This makes sense since you want to break down fatty acids for energy when you’re fasting, not use energy to
build them up from scratch. So, summing it up: to make the 16-carbon palmitoyl-CoA, we need a total of 8 acetyl-CoAs and 14 NADPH
molecules.
The other type of regulation is allosteric regulation, and that’s when a molecule increases or decreases the
activity of an enzyme by binding to a different site on the enzyme than the site where the substrate binds. Once the fatty acids are made, they’re stored in the liver and fat cells as triacylglycerides, and when they’re
needed, they can be broken down in order to make ATP.
Citrate allosterically increases the activity of acetyl-CoA carboxylase, while fatty acids allosterically inhibit it,
signaling that we have enough fatty acids and we don’t need to make more.
Summary
Alright, here comes the nitty gritty part. In order to polymerize our acetyl-CoA monomers, we need an enzyme
complex called the fatty acid synthase complex.
Alright, a quick recap. Fatty acids are energy-rich carbon compounds that are synthesized in the cytoplasm of
Overall, the enzyme complex is made up of lots of different enzymes and looks a bit like a kidney bean. liver and fat cells and are regarded to be one of the main forms of energy storage.

The complex has two separate binding domains at each end of the bean; on one end there’s an acyl carrier
protein, or ACP, and on the other end there’s an enzyme that has a cysteine amino acid in a very exposed
position.
The ACP is where acetyl-CoAs and malonyl-CoAs bind initially, and the cysteine amino acid residue is where they
hop on board the growing lipid.
Fatty acid synthesis is the conversion of 2-carbon acetyl-CoA molecules into the 16-carbon palmitoyl-CoA, in the
presence of NADPH.
The 3 major steps of fatty acid synthesis are the citrate shuttle, acetyl-CoA carboxylase, which is the rate-limiting
enzyme, and finally the fatty acid synthase complex.

It all starts, when an enzyme that’s part of the fatty acid synthase complex called acetyl-CoA ACP transacylase
removes a CoA group from an acetyl-CoA molecule.

The enzyme then attaches the 2-carbon molecule acetate to ACP, and then acetate spontaneously hops on to
the cysteine residue.

Now that acetate is bound to the cysteine residue, the ACP residue is empty.

So it’s time to bring over that initial malonyl-CoA that we made in the first step of fatty acid synthesis.

Once more, there’s an enzyme that’s part of the fatty acid synthase complex called malonyl-CoA ACP
transacylase which removes a CoA group from the malonyl-CoA molecule.

The enzyme then attaches the 3-carbon molecule malonate to the ACP group.

So now we have a 2-carbon acetate on the cysteine end, and a 3 carbon malonate on the ACP end.

At this point, another enzyme in the fatty acid synthase complex called 3-ketoacyl-ACP synthase does two
important things.

First, it cuts off the carbon that acetyl-CoA carboxylase previously added and releases it as CO2, leaving behind a
2 carbon acetate.

Then, 3-ketoacyl-ACP synthase moves this acetate and condenses it with the acetate molecule attached to the
cysteine group.

2-carbons on top of 2-carbons gives us a 4-carbon fatty acid chain attached to the cysteine group.

These two reactions require hydrogen from 2 NADPH molecules.

This cycle repeats again when the next incoming malonyl-CoA molecule attaches to the free ACP group.

And it happens for 7 cycles until we’ve got a 16-carbon long fatty acid polymer.

Per cycle, we need 1 acetyl-CoA to be converted to malonyl-CoA, and 2 NADPH molecules.

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Cholesterol metabolism
Cholesterol is a lipid molecule that helps maintain the structure of cell membranes, and is a precursor to steroid
hormones, bile acids, and vitamin D.
As it turns out, we make most of our cholesterol ourselves, but some comes through the diet.
Cholesterol synthesis, also called the mevalonate pathway, happens in the smooth endoplasmic reticulum of a
cell.
It begins with 2 acetyl-CoA molecules getting joined together by the enzyme acetyl-CoA acyl-transferase.
The result is a 4-carbon molecule called acetoacetyl-CoA and then a free CoA molecule.
Next, the enzyme HMG-CoA synthase combines acetoacetyl-CoA and acetyl-CoA to form a 6-carbon molecule
called 3-hydroxy-3-methylglutaryl CoA, or HMG-CoA - so 3 acetyls and the an free CoA molecule.
Then, an enzyme called HMG-CoA reductase reduces HMG-CoA into mevalonate, by removing a CoA-SH and a
water molecule.
This step with HMG-CoA reductase is the rate-limiting step of cholesterol synthesis.
In other words, the rate of this reaction determines the overall rate of cholesterol synthesis - it's like the slowest
step in the assembly line for a factory.
Now, cholesterol synthesis is regulated by a trio of proteins - sterol regulatory element binding protein - or
SREBP and two others that just go by SCAP and INSIG-1.
Let’s say that cholesterol levels drop because there’s less cholesterol coming into the cell from the diet.
In that situation, INSIG-1 falls off of SREBP, like pulling a pin from a grenade, and the SREBP-SCAP complex then
gets cleaved by cellular enzymes.
The cleaved SREBP floats into the nucleus, and binds to the sterol regulatory element on the DNA.
When it binds, it increases expression of the genes encoding HMG-CoA reductase.
That leads to more HMG-CoA reductase, which speeds up endogenous cholesterol synthesis.
Once HMG-CoA reductase has made the 6 carbon mevalonate, it then undergoes a number of additional
enzyme-mediated transformations before it becomes cholesterol.
First, the enzyme mevalonate-5-kinase uses adenosine triphosphate, or ATP, to phosphorylate mevalonate,
creating mevalonate-5-phosphate.
Then, phosphomevalonate kinase uses another ATP to phosphorylate mevalonate-5-phosphate, making
mevalonate pyrophosphate.
Finally, mevalonate pyrophosphate decarboxylase removes a carboxyl group from it, forming a 5 carbon
molecule called isopentenyl pyrophosphate.
Next, geranyl transferase condenses 3 of these isopentenyl pyrophosphate molecules to form a 15 carbon
molecule called farnesyl pyrophosphate.
Then, the enzyme squalene synthase condenses two molecules of farnesyl pyrophosphate to form a 30 carbon
molecule called squalene.
Squalene is pretty cool, it’s the last linear precursor to cholesterol and it’s also what helps sharks float. Yep - you
heard that right.
Okay next, an enzyme called oxidosqualene cyclase converts linear squalene molecule into a structure with rings
- a process called cyclization.
The result is our first sterol intermediate, called lanosterol.
From there, there are 19 steps of successive modifications, like molding a lump of clay into a beautiful bowl; that
convert lanosterol first into 27 carbon 7-dehydrocholesterol, and then finally into 27 carbon cholesterol!
Now - it’s worth remembering 7-dehydrocholesterol.
That’s because if it gets hit by a single photon of sunlight in just the right way, then you get cholecalciferol, or
Vitamin D3 - which is important for calcium and phosphate metabolism.
Now with regard to cholesterol - the majority is made and used by the liver - ending up as a bile acid.
There are two types of bile acids - cholic acids and chenodeoxycholic acids.
Both get conjugated by amino acids.
Conjugation with taurine makes taurocholic acid and taurochenodeoxycholic acid, while conjugation with glycine
makes glycocholic acid and glycochenodeoxycholic acid.
These bile acids are stored in the gallbladder, and released into the intestine after meals to assist in fat
digestion.
From the intestine, some bile acids are eliminated through feces - but most are reabsorbed by the intestine and
get into the portal bloodstream and back to the liver cells.
This is called enterohepatic circulation.
Now, cholesterol is insoluble in water, so moving through the bloodstream is done through lipoproteins - which
are part lipophilic - fat-loving - and part hydrophilic - water-loving.
Now, aside from the liver, every cell can make cholesterol for itself.
One way cells use cholesterol is as part of the cell membrane.
And the role of cholesterol is twofold.
At low temperatures, it squeezes in between phospholipid molecules and keeps them from packing too tightly
together to keep the membrane more fluid.
And at high temperatures, cholesterol pulls phospholipid molecules together, decreasing the space between
them.
So cholesterol makes the cell membrane fluid and durable, no matter the weather.
Finally, cholesterol can also be used by the adrenal glands and the gonads to make steroid hormones.
Enzymes in the cortex of the adrenal glands tweak the cholesterol molecules to make corticosteroids, such as
cortisol and aldosterone, which play a role in our fight or flight response, and regulating blood pressure,
respectively.
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The gonads have a different set of enzymes that metabolize cholesterol a bit differently.

The testes mostly use cholesterol to make testosterone, while the ovaries mostly use cholesterol to synthesize
estradiol and progesterone.

Summary

Alright, as a quick recap - cholesterol is made in the smooth endoplasmic reticulum of cells throughout the body.

The rate limiting step of this reaction is the reduction of HMG CoA to mevalonate which is done by HMG-CoA
reductase.

The molecules are combined to eventually form squalene - the last linear precursor in cholesterol synthesis.

Squalene undergoes cyclization to become lanosterol, and lanosterol then undergoes several modifications to
become cholesterol.

Cholesterol is mostly used in bile acids to help with fat digestion, but it also plays a role in cell membrane fluidity
and steroid hormone synthesis.

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Ketone body metabolism
In life, it's helpful to have a plan B in case plan A doesn’t work out.
In terms of energy, the body’s plan A is to generate energy from carbohydrates, fats, and proteins - basically in
that order.
But if these main fuels aren’t readily available, then plan B is to use an alternative fuel source - ketone bodies.
Ketone bodies are a group of carbon-containing molecules produced by liver mitochondria using a 2-carbon
molecule called acetyl-CoA.
The liver makes ketone bodies in physiologic states like prolonged fasting or exercise, as well as in pathological
states like type 1 diabetes mellitus or alcoholism.
Ketone bodies can be released into the circulation and get picked up by the majority of cells.
Inside the cells, they’re reconverted back into acetyl-CoA, at which point they can then enter the mitochondria
and produce ATP.
The 3 primary ketone bodies are acetoacetate, beta-hydroxybutyrate, and acetone.
Alright, so let’s say you decide to go on a 5-day fast.
About 12 hours into your fast, your blood glucose levels start to dip.
In response, glucagon is secreted from the pancreas and stimulates hepatic glycogenolysis - meaning that the
liver begins to break down glycogen into glucose and release that glucose into the blood.
About 24 hours into your fast, your liver begins running out of glycogen, so it starts the process of
gluconeogenesis which is where it makes new glucose molecules from substrates like amino acids.
Then, around 1 to 3 days into your fast, your body begins to run out of the necessary substrates to make new
glucose.
So, it switches to breaking down fatty acids for energy.
Fatty acids are mobilized from fat stores and are broken down to acetyl CoA through beta oxidation in the
mitochondria of most cells - except for brain cells.
See, thing is, fatty acids can’t cross the blood-brain barrier, so brain cells can only use glucose for energy - or,
when there’s no glucose they use ketone bodies.
This makes sense from the liver’s standpoint as well because normally, acetyl-CoA combines with oxaloacetate
in the citric acid cycle to make citrate.
But since oxaloacetate is also a substrate for gluconeogenesis, so it’s levels are pretty depleted at this point in
starvation.
So oxaloacetate basically leaves all that acetyl-CoA hanging out by itself. Not cool oxaloacetate, not cool.
This means that the liver is practically overflowing with acetyl-CoA, and the liver converts it into ketone bodies,
that various cells in our body, including the brain cells, can use.
Ketone body synthesis begins with 2 acetyl-CoA molecules getting joined together by the enzyme acetyl-CoA
acyl-transferase.
The result is a 4-carbon molecule called acetoacetyl-CoA and then a free CoA molecule.
Next, the enzyme HMG-CoA synthase combines acetoacetyl-CoA and acetyl-CoA to form a 6-carbon molecule
called 3-hydroxy-3-methylglutaryl CoA, or HMG-CoA - so 3 acetyls and then a free CoA molecule.
This step with HMG-CoA synthase is the rate-limiting step of ketone synthesis.
In other words, the rate of this reaction determines the overall rate of ketone synthesis - it’s like the slowest
step in the assembly line for a factory.
Next, the enzyme HMG-CoA lyase removes acetyl-CoA from HMG-CoA, leaving behind an acetoacetate molecule
- so two acetyls and no CoA molecules.
Acetoacetate is our first ketone body.
Acetoacetate can also be converted into another ketone body.
This happens when the enzyme beta-hydroxybutyrate dehydrogenase takes hydrogens from a nicotinamide
adenine dinucleotide, or NADH molecule and gives them to acetoacetate, forming beta-hydroxybutyrate, our
second ketone body.
However, acetoacetate can also enter the bloodstream.
At this point some acetoacetate molecules remain in that form, and some acetoacetate molecules
spontaneously lose a carbon - no enzymes required - to create acetone, our 3rd ketone body.
But this acetone is metabolically useless to cells, so it’s simply exhaled from the lungs.
That’s why in pathological states like diabetic ketoacidosis, the breath smells a bit like fruit - that’s the smell of
acetone.
Now, these ketone bodies are acidic molecules that like to donate their protons, so they can lower the blood pH,
causing a metabolic acidosis.
Once beta-hydroxybutyrate and acetoacetate are in the bloodstream, they can diffuse into the mitochondria of
peripheral tissues, like the brain, skeletal muscle and kidneys.
One cell that can’t use ketone bodies is the red blood cell, because it doesn’t have mitochondria.
Initially, beta-hydroxybutyrate is converted back to acetoacetate by the same enzyme, beta-hydroxybutyrate
dehydrogenase.
However this time, the enzyme takes hydrogen from beta-hydroxybutyrate and gives it to NAD+, making NADH.
Next, acetoacetate needs a CoA molecule to be converted to acetoacetyl-CoA.
But unfortunately, peripheral tissues don’t have a lot of acetyl-CoA, so the enzyme thiophorase grabs it from
succinyl-CoA, resulting in acetoacetyl-CoA and succinate.
Ironically, liver cells don’t have this thiophorase enzyme, so they can’t use ketone bodies for energy, they can
only make them for other cells.
A great example, of how the liver is the humble servant of the body.
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Finally, an enzyme called beta-ketothiolase cleaves acetoacetyl-CoA, and then adds an extra CoA, making 2
acetyl-CoA molecules, which can then enter the citric acid cycle to make ATP.

Summary

Alright, a quick recap. Ketone bodies like beta-hydroxybutyrate and acetoacetate are an alternative source of
energy in states of prolonged starvation.

Ketone body synthesis occurs in the liver, where excess acetyl-CoA from fatty acid breakdown is used as a
substrate.

Once synthesized, ketone bodies can leave the liver, and enter into peripheral cells such as the brain, skeletal
muscle and kidney, where they can be reconverted back into 2 acetyl-CoA molecules that can be used to make
ATP.

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Amino acids and protein folding
Proteins are vital for the normal function of a cell.
Essentially, a protein is, at its simplest, a very long chain of individual units, called amino acids, bound to each
other by peptide bonds to form an amino acid chain.
They sorta resemble a string of beads, and they get twisted and folded into a final protein shape.
To make a protein, we need to get to know two things - the “ingredients”, which are the amino acids, and the
“recipe” - or how the finished amino acid chain folds into the protein.
Humans use 20 amino acids in our day-to-day protein making.
Let's get to know them a bit better. So, we have: alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid
(Asp), cysteine (Cys), glutamic acid (Glu), glutamine (Gln), glycine (Gly), histidine (His), isoleucine (Ile), leucine
(Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan
(Trp), tyrosine (Tyr), valine (Val). Phew, that’s 20.
One way to divide them, is into the ones that we make ourselves, and the ones that we cannot.
There are 5 amino acids that are dispensable - alanine, aspartic acid, asparagine, glutamic acid, and serine -
because we can make them de novo ourselves at any time, and in good quantity.
Then, there’s 6 of them that we call conditionally essential because we can make them most of the time, but not
always - arginine, cysteine, glutamine, glycine, proline, and tyrosine.
Finally, there are 9 of them that we cannot make ourselves - His, Ile, Leu, Lys, Met, Phe, Thr, Trp, and Val, and as
a result we have to obtain them from our diet. We call these the essential amino acids.
Okay, so, the amino acid. Just from the name, you can tell they’ve got an amine group, or “NH2”, and also an
acid, in this case a carboxylic acid group “COOH”.
The amine and carboxylic acid groups are both bound to the same carbon, called the alpha carbon.
Now, at a physiologic pH of 7.4, the amine group has a positive electrical charge, and the carboxyl group has a
negative charge.
Having both a positive and a negative charge makes amino acids a type of zwitterion - which is German for
“hybrid”, or “double ion”.
Now, the alpha carbon also has a side chain, sometimes marked as “R”.
And this side chain gives the amino acid certain properties, which can play an important role in the overall
protein structure.
First the side chain can be hydrophilic or hydrophobic - so water loving or water hating. Hydrophobic amino
acids have nonpolar side chains.
This might be in the form of an alkyl side group, which is a saturated hydrocarbon, seen in valine, glycine,
alanine, leucine, isoleucine, methionine, and proline.
Alternatively, it might be in the form of an aromatic side group - which involves a 6-carbon ring, like in
phenylalanine, tyrosine, and tryptophan.
Now, hydrophilic amino acids have polar side chains.
These polar side chains might be acidic - like when their side chains contain additional carboxyl -COOH groups,
like aspartic acid and glutamic acid.
Other hydrophilic amino acids have polar side chains that are basic, like lysine, histidine, and arginine.
At physiological pH the acidic groups lose a hydrogen and the basic groups gain a hydrogen.
Finally, some polar side chains are neutral, for example they can contain hydroxyl groups, -OH, like serine,
threonine, or tyrosine, or sulfhydryl groups -SH, like cysteine, or carboxamide groups (R-C=0-NH2) like
asparagine or glutamine.
Now, keep in mind that the charge on an amino acid really depends on its side chain as well as the pH.
For example, at a very low pH, the amine group is positive, while the carboxyl group is neutral.
And at a very high pH, the amine group is neutral, while the carboxyl group has a negative charge.
And at a pH that’s somewhere in between, both groups are electrically charged and they cancel each other out,
resulting in no net charge for the amino acid.
The “just right” pH, also known as the pI, or isoelectric point, is different for every amino acid, and it depends on
the specific side chains.
For amino acids to link up in a chain, the carboxylic -COOH group of one amino acid has to bind to the amine -
NH2 group on another amino acid, creating a single peptide bond.
This is a condensation reaction - meaning that two amino acids are basically smushed together, and the OH from
the carboxyl group, along with one of the hydrogens from the amine group, get released as a water molecule in
the formation of an amide bond.
While technically being a single bond, it actually has the properties of a structurally stronger double bond,
thanks to the property of resonance.
Now, resonance is a property of a molecule where electrons get shared across the molecule, while keeping the
arrangement of atoms the same.
Basically, the electrons from neighboring functional groups in the amino acid are “borrowed”, and that makes
peptide bond stronger and more stable.
So, amino acids are, essentially, a carboxylic -COOH group, an amine -NH2 group, a side chain, and a hydrogen
bound to an alpha carbon.
Now, an interesting geometric property called chirality - means that the same amino acid can exist in two forms,
that look like mirror images of each other.
These two forms are called enantiomers of one another.
We have the left, or levo-oriented amino acids, as well as the right, or dextro-oriented amino acids.
While similar, they are definitely not the same. Think of shoes. While made out of the same materials, and
generally looking alike, the left and the right shoe are not interchangeable, at least not without a lot of pain
involved. As it turns out, proteins are made out of only L-amino acids.
Now, protein production itself happens in cellular structures called ribosomes, which use the messenger RNA
which is essentially a blueprint that tells the ribosome exactly the order of amino acids that are needed.
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At this point, the protein is just a growing string of amino acids - as it grows, it’s either being injected into Quaternary structure is the final level, and it’s the level at which multiple polypeptide chains come together to
another organelle, called the endoplasmic reticulum, which will help the protein take shape or it’s being form a larger protein structure.
translated directly into the cytosol.
A classic example involves the 4 polypeptide subunits that have to come together to form a single hemoglobin
Now, the proteins have multiple levels of structure to them - primary, secondary, tertiary, and quaternary protein, which is, roughly, a tetrahedral arrangement.
structure, creating a hierarchy.

As an analogy, think of the alphabet. It can be used to create words, which can make simple sentences, which
can further be made into complex sentences.
As an example, the letters themselves would be considered the primary structure “I N E X A M I S T H E H O U R S
T W O”.
Then simple words like “EXAM” and “HOURS” would represent secondary structures.
Tertiary structure would be when the entire chain folds together, perhaps making a simple sentence like “THE
EXAM IS IN TWO HOURS!”
And the quaternary structure might actually be a few peptide chains coming together to form a more complex
protein, making a complex sentence that says “THE EXAM IS IN TWO HOURS AND I HAVEN’T SLEPT AT ALL”.
Summary
Alright, as a quick recap. There are 20 amino acids - 5 dispensable, 6 conditionally essential, and 9 essential.
The primary structure of a protein is the linear sequence of amino acids.
The secondary structure includes both α-helix or β-pleated sheets, both of which rely on hydrogen bonds.
The tertiary structure binds the secondary structures through various other bond interactions like disulfide
bridges or hydrophobic reactions, and, the quaternary structure creates the final shape of a protein by
connecting multiple polypeptides in the form of tertiary structures.

When it comes to proteins, the primary structure is simple enough - it’s just a linear sequence of amino acids
connected through peptide bonds - like a string of pearls.

Now, the peptide bonds between the amino acids are very rigid, but by comparison the single bonds connecting
the amide functional group of the peptide bond to the the alpha carbon are flexible.

That allows significant freedom of rotation, and through that rotation, the protein can fold into one of two types
of secondary structure - α-helix or β-pleated sheets.

The α-helix resembles a spring.

The helical structure brings the C=O of the first amino acid near the N-H of the fifth amino acid, the second C=O
gets near the sixth N-H, and so on. In other words: each of these instances is separated by 4 amino acids.

Having the O and H get close to one another allows for a strong hydrogen bond to form, and that makes the α-
helical structure really stable!

β-pleated sheets also rely on hydrogen bonding - but slightly differently.

Imagine a neatly folded piece of paper. In β-pleated sheets, hydrogen bonds form between the N-H on one flap
of paper and the C=O on another flap of paper, and these bonds almost hold or “glue” the sheets together.

That makes beta-sheets really stable as well.

Now, tertiary structure is the overall shape of the polypeptide chain, and it includes the secondary structures as
well as other features.

For example, two sulfur containing cysteines, can bind to form a disulfide bridge.

Also, hydrophobic amino acids form bonds with one another and orient themselves towards the inside of the
protein, so that they’re able to avoid contact with water.

It’s as if the hydrophobic amino acids are being a bit shy.

Basically, the way a polypeptide chain twist and turns to form its tertiary structure is a bit like the way
headphones get tangled up in your pocket.

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So enzymes decrease that extra energy requirement for the reaction - graphically turning our mountain into a
hill.
Enzyme function Consider this analogy. Imagine a little boy who’s nervous about getting a vaccine - he’s the substrate, and he
turns into a vaccinated child - that’s the product.
Enzymes are proteins that play a major role in the biochemical reactions happening every moment inside our The transition state is where the needle goes in, and as you can imagine - the boy might get really anxious and
bodies - everything from digesting a bowl of ramen noodles to flexing your muscles in front of a mirror. upset - a highly energetic and uncomfortable state.
Enzymes act as catalysts - meaning that they speed up the rate at which these biochemical reactions happen. In this scenario, enzymes are like adults who hold the boy and calm him down, reducing the anxiety or energy
level of the transition state and making the whole thing happen faster.
So instead of waiting months to years for a reaction to happen, it can happen in seconds - which is essential for
life to happen. Fortunately, enzymes don’t get used up in the process. They attach to the substrate until it turns into the
product and then release the product.
Imagine trying to digest a single bowl of ramen for a year - you'd die of hunger before you could do it!
As soon as they’re done, they find another substrate.
Every biochemical reaction has a substrate and a product - so let’s put them on this graph called a reaction
coordinate diagram. What’s more is that enzymes and substrates are like biochemical soulmates - each enzyme is specifically
designed for a particular type of substrate.
The X axis shows how a reaction progresses, while the Y axis shows the energy level at the different points along
the reaction. For example, amylase is an enzyme in your saliva that specifically helps break down large carbohydrates - into
smaller sugar molecules that are then further broken down by other enzymes.
In the beginning, we’ve got the substrate - let’s call it A - with a fair amount of free energy.
Now, the rate at which enzymes catalyse biochemical reactions is called enzyme kinetics, and there are two
At the end of it, there’s the product - or B, which ranks lower energy-wise.
graphical ways to look at this.
The energy of the product minus the energy of the substrate is called the energy of the reaction, also known as
The first, is the Michaelis Menten graph which has the concentration of the substrate, or [S], on the X axis, and
Gibbs free energy, or ΔG.
the speed, or velocity of the reaction or V, which is how much product is formed over time, on the Y axis.
Because lower energy states are preferred, a reaction spontaneously occurs when the product has a lower free
If there’s a fixed amount of enzyme, the velocity of the reaction increases as more substrate is added - that is,
energy than the substrate - so a negative ΔG.
until all the active sites on all of the enzyme become saturated.
So let’s say we’re looking at one such spontaneously occurring reaction, but between going from the substrate
At this point, adding more substrate won’t do a thing, because there’s no more enzyme to bind it - so the speed
to the product there’s an intermediate transition step that has a really high energy state.
of the reaction plateaus.
The amount of extra energy the substrate requires to get to the transition state - so the height of the upslope - is
The point where the curve flattens out corresponds to the maximum velocity, or Vmax, on the Y axis.
called the activation energy - or ΔG‡.
Now we can determine Km - which the concentration of substrate at which the speed of the reaction is exactly
As soon as it enters the transition state, the molecule is highly unstable - and wants to go to a more stable
half the maximum velocity.
lower-energy molecule.
So we look at the Y axis, find what half of Vmax is, then we go parallel to the X axis until we reach our reaction
It either goes back to being a substrate or to being a product.
curve.
If it’s a substrate once again, it can go back up to the transition state if there’s enough activation energy once
From there, we go straight down towards the X axis - and Km will be equal to that substrate concentration.
more, but if it becomes a product then it needs even more energy to get back to the transition state.
The reason that Km is worth figuring out is that it inversely reflects enzyme affinity - if Km is low, only a little
That’s why over time, with millions of molecules doing this, the majority of substrate turns into product.
substrate is needed for the reaction to skyrocket up to half of its maximum rate, so we’re looking at an enzyme
Now, without an enzyme, the substrate might eventually harness enough activation energy to enter the with high affinity.
transition state - but enzymes help speed things up quite a bit.
On the other hand, if Km is high, then it takes a lot of substrate to get the reaction to go at half the maximum
Enzymes are proteins that are folded in a particular way, so that they have a pocket called the active site on their rate - so the enzyme has low affinity for its substrate.
surface.
Fundamentally, these graphs tell us about binding.
When enzymes get involved in a reaction, the substrate binds to the active site, and together they form an
In fact, the same graphic representation could be used to show how strongly drugs binding to receptors, or even
enzyme-substrate complex, and that helps stabilize the transition state.
transcription factors binding to DNA!

So while we’re here, let’s look at how Vmax or Km can increase or decrease.

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Vmax depends on the number of enzymes that can catalyse the reaction - so increasing or decreasing the Y intercept is equal to 1/Vmax, and X intercept - since it happens negative to the origin of the axes - is -1/Km.
number of enzymes will increase and decrease Vmax.
Thus, the x- and y-intercepts can be used to quickly identify Km and Vmax.
One way to increase Vmax is induction, which is when there’s an increase in gene expression of an enzyme.
The only trick is that, much like our reciprocal equation, now everything is upside down! So processes like
The opposite - a decrease in Vmax - might happen if there’s repression or silencing of gene expression. induction and upregulation which increase the Vmax, actually cause 1/Vmax to decrease so the line representing
Km/Vmax would slope lower on the graph than the control.
If we’re talking about membrane receptors or transporters, up-regulation or down-regulation of receptors or
transporters on the cell’s surface would cause an increase or decrease in Vmax, respectively. Conversely, processes like repression or downregulation or even non competitive inhibition which decrease the
Vmax, will increase 1/Vmax, making the line slope higher on the graph than the control.
Finally, a drug might block enzymes, transporters or receptors non-competitively and cause a decrease in Vmax.
So interpreting a Lineweaver Burke plot goes like this.
With non-competitive inhibition, the inhibitory molecule binds irreversibly to the enzyme active site, or
reversibly to a different place called the allosteric site. Stimulation makes the straight line appear to go down, inhibition makes it appear to shoot higher - it’s, well, the
reciprocal!
The net effect is that substrates can no longer bind to the active site and it is as if the number of available
enzymes had decreased.
Summary
So the effects of non-competitive inhibition are similar to what happens when there’s less enzyme around,
decreasing Vmax.
Alright, as a quick recap. Enzymes lower the activation energy required for the substrate to enter the transition
Now let’s look at Km, which varies according to enzyme affinity. state.

Affinity depends on the shape of the enzyme, and how well the substrate fits into the active site.
So, let’s say we alter the enzyme in a way that makes it bind more substrate - one way to do this is when a
molecule called an activator binds to the enzyme and increases that enzyme’s affinity for the substrate, lowering
the Km. Conversely, competitive inhibition can be used to decrease the affinity of the enzyme.
Substrate binds to the enzyme at the active site, forming an enzyme-substrate complex, and it is released after
it’s transformed into the product.
There are two main ways to look at enzyme kinetics: the Michaelis Menten graph, and the Lineweaver Burke
plot.

A competitive inhibitor binds to the active site instead of the substrate, and doesn’t get metabolized - blocking Both can be used to determine the two kinetic variables, Vmax, or the maximum speed of the reaction, and Km,
the enzyme. or the affinity of the enzyme for its substrate.

Now, since this is a reversible process, adding more substrate means that it’s possible to eventually outcompete
and displace the inhibitor. But this increases the Km.

Now, practically speaking, a common way to change the shape of an enzyme is through phosphorylation by
kinases or dephosphorylation by phosphatases, and many processes are regulated this way in our cells.

In fact, the terms desensitization and sensitization refer to changes in a receptor’s shape which decrease or
increase its affinity for a drug.

Now, let’s switch gears and look at the Lineweaver-Burke plot.

This graph is based on the Michaelis Menten equation - which states that the initial speed of the reaction - or V0
- is equal to the concentration of substrate - [S] times the maximum velocity - Vmax, over the concentration of
substrate + Km.

So here’s where things get interesting - to get to our Lineweaver Burke plot, we’re writing this equation down as
a double reciprocal.

So flipping the whole thing upside down, we get that 1 over V0 is equal to Km over Vmax plus 1 over Vmax plus 1
over the concentration of substrate - [S].

Now we can use this equation on the plot: 1/V0 goes on the Y axis and 1/[S] goes on the X axis.

Conveniently enough, after flipping the equation upside down, Km/Vmax is represented as a straight line, that
intercepts both the Y and the X axes.

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Amino acid metabolism
Amino acids are the building blocks of proteins.
And just like how you can make lots of words with a finite alphabet, it's possible to make lots of proteins
with just 20 amino acids!
Each amino acid has nitrogen-containing amine group, and a carboxylic acid - hence the name amino acid!
Each amino acid also has a unique side chain that’s kind of like the amino acid’s fingerprint.
10 of the 20 amino acids are essential, meaning that you obtain them from dietary sources rich in protein,
such as meats or tofu.
The other 10 amino acids are non-essential, which means that they can be made in our body, so you don’t
have to get them from your diet.
So let’s say you had a nice big bowl of lentils rich in protein.
Protein would get broken down into amino acids, and those amino acids would make their way into various
cells to serve as building blocks in protein synthesis.
And the cell has to try to make use of these amino acids, because ammonia which is the nitrogen-containing
amine group in amino acids, can become toxic to the cell if it gets freed up and starts to build up in the cell.
Ultimately, to get rid of it, ammonia must first be removed from the amino acid and then sent to the liver
where it can get metabolized into a less toxic molecule called urea.
To do that, a group of enzymes called transaminases or aminotransferases transfer that nitrogen containing
amino group from amino acids to ketoacids, like alpha-ketoglutarate.
These reactions are called transamination reactions and they’re reversible reactions, meaning that the
reaction can go in either direction using the same enzyme.
And generally speaking, transamination reactions requires pyridoxine, or vitamin B6, as a cofactor to help
move things along.
So let’s take an example of a transamination reaction with the amino acid alanine in a muscle cell.
First, the enzyme alanine transaminase, or ALT, switches the amino group on alanine with the oxygen
group on alpha-ketoglutarate, resulting in a ketoacid called pyruvate, which now has the oxygen group, and
the amino acid glutamate, which now has the amino group.
Now pyruvate has two options. First, it can be converted into acetyl-CoA by pyruvate dehydrogenase in the
muscle cell, and then the acetyl-CoA can then enter the citric acid cycle.
Second, pyruvate can be converted to lactate by lactate dehydrogenase, and lactate can then travel to the
liver.
Because this is a reversible reaction, lactate can be reconverted back to pyruvate in the liver by lactate
dehydrogenase as well.
Within the liver, pyruvate can enter gluconeogenesis, and help form a new glucose molecule.
Glucose can then enter the circulation, and go back to the muscle cell, where it can be broken down in
glycolysis to make ATP and our original friend, pyruvate.
This nifty little cycle is called the Cori cycle.
Now glutamate is a unique amino acid, and it’s the only amino acid that doesn’t have to transfer its
nitrogen-containing amine group to another molecule.
That’s because glutamate undergoes oxidative deamination, a process that removes hydrogens and an
amino group.
So specifically, what happens, is that glutamate travels to the liver mitochondria.
This process takes place in the mitochondria because free ammonia in the cytoplasm can damage the cell.
So within the relatively safe environment of the mitochondria, the enzyme glutamate dehydrogenase
removes glutamate’s amino group and adds an oxygen group from water - forming alpha ketoglutarate.
At the same time, glutamate’s hydrogens get transferred to nicotinamide adenine dinucleotide, or NAD+,
forming NADH.
The free ammonia in the mitochondria, can then be converted into the less toxic molecule urea in a process
called the urea cycle.
And remember that all of these reactions are reversible.
So when the body needs amino acids, these reactions can go in full reverse using the same enzymes to
synthesize our original amino acid - alanine.
Now in addition to the nitrogen-containing amine group, the cell still has to metabolize the rest of the
amino acid.
Some amino acids such as alanine and glycine, serve as substrates to make new glucose, we call those
glucogenic amino acids.
Other amino acids, such as leucine and lysine form alternative energy fuels called ketone bodies, and we
call those ketogenic.
And other amino acids, such as valine and aspartate can feed directly into the citric acid cycle, and lead to
the formation of energy as ATP.
Some amino acids such as phenylalanine can do it all, being glucogenic, ketogenic and feeding into the citric
acid cycle as well.
Summary
Alright, a quick recap. Amino acids have a nitrogen-containing amine group that has to be safely
metabolized to avoid toxicity.
To do that, cells use transamination reactions with a ketoacid such as alpha ketoglutarate to generate
intermediates like pyruvate and glutamate.
Glutamate can then be oxidatively deaminated in the liver mitochondria to get rid of ammonia in the urea
cycle.
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Biochemistry-metabolism

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Biochemistry-metabolism
Nitrogen and urea cycle
The human body generates a lot of waste products, and fortunately, our kidney is capable of getting rid of most
of them.
However, there is one arch nemesis that the kidney can't deal with on its own. So, the liver comes to the rescue.
The villain is ammonia.
Ammonia is the major toxin that results from the metabolism of amino acids.
Amino acids are made of a nitrogen group, a carbon skeleton, and a side chain that is unique to each amino acid.
When amino acids are metabolized, the nitrogen is formed into ammonia, and ammonia is toxic to the cells.
So the ammonia is shuttle over to the liver and sent through the urea cycle, which is a series of enzymatic
reactions that convert ammonia into urea.
The urea cycle takes place within the mitochondria, so that it doesn’t affect proteins and organelles in our
cytoplasm.
Urea can then easily be dealt with by the kidney.
It’s a bit like how a mama bird might mash up a worm so that it’s easier for a baby bird to digest.
In this case the liver is the mama bird, and the kidney is the baby bird.
Alright, so first, ammonia needs to get to the liver.
And it has to be done carefully because it’s toxic.
So, much like a prisoner, it needs to be carried in the circulation by a police officer, to its prison, which is the
liver mitochondria.
There are two ways this can happen. The first way is used throughout by cells throughout the body.
The enzyme glutamine synthetase adds ammonia to the amino acid glutamate forming glutamine.
Glutamine can move into the blood, and essentially transport ammonia around the block, until it gets to a liver
cell.
Once inside the mitochondria of a liver cell, an enzyme called glutaminase cleaves glutamine back into
glutamate and ammonia, and the ammonia can then enter the urea cycle.
The second way to move ammonia around is done mostly by skeletal muscle cells.
In skeletal muscle cells, the enzyme glutamate dehydrogenase incorporates ammonia into the molecule alpha-
ketoglutarate and turns it into glutamate.
But unlike glutamine, glutamate can’t leave the cell on its own.
It needs to somehow give its ammonia to an amino acid that can leave the cell, and that’s alanine.
So, the enzyme alanine transaminase, or ALT, converts glutamate and pyruvate into alpha-ketoglutarate and
alanine.
And alanine then moves into the blood, and ends up transporting ammonia to a liver cell, and once there it
undergoes the reverse of the previous reactions.
The enzyme alanine transaminase converts alanine and alpha-ketoglutarate back to pyruvate and glutamate.
At this point, the ammonia is now part of glutamate once again.
Okay, so now these two pathways both end up at the same point in the liver cell, which is in the form of
glutamate.
From there, glutamate has two possible outcomes. The first is for glutamate to encounter the same enzyme that
incorporated ammonia into glutamate; glutamate dehydrogenase, and to have the ammonia snatched away
from glutamate, converting it back into alpha-ketoglutarate and a free ammonia the enters the urea cycle.
And since this reaction can happen in both directions, we call it a reversible reaction.
The second outcome is for glutamate to encounter the enzyme aspartate transaminase, or AST, and combine
with oxaloacetate to form aspartate and alpha-ketoglutarate.
As before, the amino acid aspartate is carrying the ammonia group, and aspartate will directly enter the urea
cycle - in fact, it’s the only amino acid to do that!
Alright, now that we have ammonia in the liver, the urea cycle can begin.
The urea cycle is made of a series of enzymatic reactions that modify the chemical structure of substrates within
it until it ultimately results in the molecule urea.
Urea is made of 2 nitrogen groups and 1 carboxyl group.
Think of the enzymes involved in the urea cycle as children playing around with clay, changing its shape until the
clay looks like urea. Sounds like fun!
Okay so the first two steps of the urea cycle take place in the mitochondria, and the rest happens in the
cytoplasm.
To kick things off, the participants in the first reaction are ammonia, carbon dioxide, and 2 adenosine
triphosphate, or ATP molecules.
An enzyme called carbamoyl phosphate synthetase 1, or CPS1 converts these into a molecule called carbamoyl
phosphate.
If we breakdown the name of this molecule, “carb” refers to the carboxyl group provided by carbon dioxide, “-
amoyl” refers to the nitrogen group provided by ammonia, and phosphate refers to the phosphate group that
comes from one of the two ATPs molecules.
Now, this process can be sped or slowed down based on the affinity of carbamoyl phosphate synthetase 1 for
ammonia.
A molecule called N-acetylglutamate allosterically activates CPS1, meaning that it binds to the CPS1 on a site
different from where ammonia binds.
When it binds, N-acetylglutamate modifies the physical shape of CPS1, so that it can process ammonia more
efficiently, increasing the rate of urea synthesis.
Now N-acetylglutamate itself is made by a mitochondrial enzyme called N-acetylglutamate synthetases which
combines glutamate and acetyl-CoA.
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And having enough N-acetylglutamate around is crucial because without it, CPS1 has very low affinity for
ammonia.

In fact, having a genetic deficiency in N-acetylglutamate can lead to ammonia building up to toxic levels.

In the second step of the urea cycle, also in the mitochondria, the enzyme ornithine transcarbamoylase
combines the molecule ornithine combines with the carbamoyl part of carbamoyl phosphate to form citrulline,
releasing free phosphate in the process.

In fact, deficiency of ornithine transcarbamoylase is the most common genetic cause of increased ammonia
levels, or hyperammonemia.

Next, citrulline goes from the mitochondria to the cytoplasm, where it meets up with aspartate.

The enzyme argininosuccinate synthetase uses the energy of an ATP molecule to combine citrulline with
aspartate to make argininosuccinate.

So now aspartate has provided the 2nd and final nitrogen group which will be part of the final urea structure.

But we have to continue playing with clay until we get to urea.

Next, the enzyme argininosuccinate lyase breaks up argininosuccinate into two molecules - fumarate and
arginine.

Let’s start with fumarate. Fumarate is first converted to malate , and then the enzyme malate dehydrogenase
converts malate to oxaloacetate.

Oxaloacetate and glutamate are converted to aspartate and alpha-ketoglutarate by the enzyme aspartate
transaminase, or AST.

This regenerates aspartate, so that it may enter the next urea cycle.

Next there’s arginine. Arginine is broken down by arginase to make urea and ornithine.

Urea exits the liver cell, enters the blood, and gets excreted by the kidneys.

Whereas ornithine heads the other way and enters the mitochondria, so that it may enter the next urea cycle.

Summary

Alright, a quick recap. All amino acids with the exception of aspartate are metabolized into a nitrogen containing
toxic compound called ammonia.

In liver cells, aspartate and ammonia provide the 2 nitrogens necessary to make the less toxic urea in the urea
cycle.

In the liver mitochondria, carbomoyl phosphate synthetase 1 requires allosteric activation from N-
acetylglutamate to bind ammonia.

Once urea is formed, it can go into the bloodstream and get excreted by the kidneys.

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Biochemistry-metabolism
Nucleotide metabolism
Nucleotides are the building blocks of nucleic acids - deoxyribonucleic acid, or DNA - and ribonucleic acid, or
RNA.
The most basic structure of the nucleotide can be broken down into three subunits - a five carbon sugar, a
phosphate group, and a nitrogenous base, also known as nucleobase.
So, the five carbon sugar is either deoxyribose or ribose - and depending on which is used, the final product is
either deoxyribonucleic acid, or ribonucleic acid.
The nucleobases can be either pyrimidines or purines.
There are 3 pyrimidine bases, and they are all made up of a single heterocyclic ring - cytosine, or C, thymine, or T
- which is DNA-specific, or T, and uracil, or U, which is RNA-specific.
There are two purine bases, adenine, or A, and guanine, or G, and they're made up of two rings.
Now if we link up just the sugar and the nucleobase, we’ve got ourselves a nucleoside.
To make a nucleotide, all we’ve got to do is add a phosphate group to the 5th carbon of the sugar on a
nucleoside.
So, nucleosides have slightly different names - in RNA, ribose plus adenine makes adenosine, guanine makes
guanosine, cytosine makes cytidine, and uracil makes uridine.
So, adding a phosphate, the “full name” of RNA nucleotides would actually be adenosine monophosphate, or
AMP, guanosine monophosphate, or GMP, cytidine monophosphate, or CMP and uridine monophosphate - or
UMP.
For DNA, we’re using deoxyribose instead of ribose, so the nucleosides would be deoxyadenosine,
deoxyguanosine, deoxycytidine and deoxythymidine - and similarly, with the addition of phosphate group, the
nucleotide would be called, for example, deoxyguanosine monophosphate, or dGMP.
We know, all of this sounds complicated. Don’t shoot the messenger.
There are two ways our cells can make nucleotides - one is to make from scratch, also known as de novo
synthesis, and the other is the salvage pathway, that recycles nucleotides that are already semi-degraded.
Let’s begin with the ribose-containing nucleotide synthesis.
De novo synthesis starts with ribose-5-phosphate for both purine and pyrimidine bases.
Ribose-5-phosphate comes from another intracellular metabolic pathway called the pentose phosphate
pathway.
And an enzyme called ribose phosphate pyrophosphokinase uses an adenosine triphosphate - or ATP - molecule,
and removes two phosphate groups from it, attaching them to to ribose-5-phosphate, creating a phosphoribosyl
pyrophosphate - or PRPP. We’ll need this later on.
Next step is to make pyrimidines.
We’ll need the amino acid glutamine, some bicarbonate, water, and ATP.
An enzyme called carbamoyl phosphate synthetase II will then create carbamoyl phosphate which is joined to
aspartate by the enzyme aspartate transcarbamoylase - or ATCase, for short.
Together, they form a ringed molecule called carbamoyl aspartic acid, which gets dehydrated by dihydroorotase
to create a molecule called orotate.
Next, the enzyme orotate phosphoribosyltransferase moves the phosphoribose unit from PRPP to orotate and
that forms orotidine monophosphate, or OMP.
Next, the enzyme UMP synthase converts orotidine monophosphate into uridine monophosphate, or UMP.
That UMP gets phosphorylated twice by nucleoside diphosphate kinase, to become uridine triphosphate, or UTP.
Finally, the enzyme CTP synthase, converts uridine triphosphate into cytidine triphosphate, or CTP.
And before CTP is used as a nucleic acid, it serves as an energy source in other cellular reactions and thereby
loses two phosphates.
Purine synthesis is a bit more complex.
It starts with the amino acids glutamine, aspartate, and glycine, together with bicarbonate and formate, which is
the anion derived from formic acid.
These undergo a ten-step pathway with the help of a number of enzymes, with names that will assure you a
victory in Hangman.
The result of all this is inosine monophosphate, or IMP, which is sort of a generic purine.
IMP is converted to AMP in two steps.
First, an enzyme called adenylosuccinate synthase uses energy from a GTP molecule to add aspartate to IMP,
forming adenylosuccinate.
Then, another enzyme called adenylosuccinate lyase cleaves adenylosuccinate into AMP and a fumarate
molecule.
Alternatively, IMP can become GMP.
To do that, IMP gets oxidized by IMP dehydrogenase to become xanthosine monophosphate, or XMP.
Then, an enzyme called GMP synthase transfers an amino group from glutamine to xanthosine monophosphate,
making GMP.
Okay, so now that we have RNA nucleotides, making DNA ones is relatively straightforward.
RNA nucleotides are usually in the monophosphate form.
For this metamorphosis to happen, we need them in diphosphate form, so CDP, UDP, ADP, and GDP.
Next, an enzyme called ribonucleotide diphosphate reductase will reduce the ribose within them into
deoxyribose, creating dCDP, dUDP dADP, and dGDP.
After this, they just need to lose a phosphate group, and we’ll have dCMP, dUMP, dAMP, and dGMP.
But, something is missing - dTMP. Making this is also fairly simple. dUMP gets converted by thymidylate
synthase into dTMP, and at that point we’re all set to make DNA.
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Biochemistry-metabolism
Now, pyrimidine rings - C, T and U - can be degraded completely back down to carbon dioxide -CO2 - and
ammonia - NH3, which can then be excreted through exhalation from the lungs and into urine.
In contrast, purine rings, or G and A - cannot be broken down in quite the same way.
Instead, they’re degraded down to the metabolically inert uric acid which is then excreted into urine.
They are produced within the cytosol, either through salvage pathways, or created anew through their
respective pathways - for pyrimidines, we need amino acid glutamine, aspartate, some bicarbonate, water, and
ATP, and for purines, we need amino acids glutamine, aspartate, and glycine, together with bicarbonate and
formate.

For GMP to become uric acid, the enzyme purine nucleoside phosphorylase, first removes the ribose and the
phosphate from it, turning it into guanine.

Next, another enzyme called guanase removes an amine group turning guanine into xanthine.

Finally, xanthine is oxidized into uric acid by the enzyme xanthine oxidase.

On the other hand, for AMP to become uric acid, first the enzyme AMP deaminase removes an amine group
from it, turning it into IMP.

Then purine nucleoside phosphorylase comes in and removes the phosphate and the ribose from IMP, making
hypoxanthine.

Hypoxanthine is then oxidised twice by xanthine oxidase - first to become xanthine, and then finally, to uric acid.

While uric acid is metabolically inert, an issue may arise if it builds up in the body - which can happen either
because of excess production, or slow removal.

When there’s too much uric acid, it gets into tissues, like joints or tendons, and can become monosodium urate,
which is a salt - and salts like to crystallize.

So crystals start forming in the tissues, and in response the body starts to secrete proteins around them - and
the protein covered crystals are called tophi.

These tophi cause an inflammatory reaction called gout - which most often involves the joint in the big toe.

Now, it turns out that those intermediate molecules in purine degradation, guanine and hypoxanthine, can be
restored into fresh new nucleic acids, through what is known as a salvage pathway.

The enzyme hypoxanthine- guanine phosphoribosyl transferase, or HGPRT for short, returns ribose and
phosphate back to guanine to form GMP, and to hypoxanthine to form IMP.

From there, IMP can become AMP again, following the AMP synthesis pathway from before.

A genetic disease, called Lesch-Nyhan syndrome is characterized by a complete absence of HGRPT.

And as a result, there’s too much uric acid getting produced by purine degradation.

Since the uric acid can’t get recycled, and our excretion is kinda slow to begin with, it begins getting stored up in
tissues, causing gout.

Lesch-Nyhan causes a number of symptoms like self-mutilation, and one distinctive one is that it causes
symptoms of gout in very young patients, which is highly unusual - so another name for it is juvenile gout.

Summary

Alright, as a quick recap. The nucleotide is a building block of DNA or RNA, and it’s made out of three parts - the
phosphate group, five carbon sugar, and a nucleobase.

The nucleobases are divided into purines, adenine and guanine, and pyrimidines, cytosine, thymine, and uracil.

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