FUNDAMENTALS HOUR 2
AUGUST 31, 2010
DEIVANAYAGAM
SYNTHESIS AND DEGRADATION OF NUCLEOTIDES
Scribe: FARAH BUTT
Proof: CALVIN SIMS
Page 1 of 5
I. Synthesis and Degradation of Nucleotides [S1]
II. Information Transfer in Cells [S2]
a. Nucleotides are one of the most important constituents of the cell because they have all of the information
recorded in them.
b. Information is stored in them and there is a beautiful way in which it is retrieved.
c. Replication, transcription, translation.
d. In some cases RNA is involved, in others DNA is involved.
III. Purines and Pyrimidines [S3]
a. If it’s a one-syllable word, it is going to be a bigger ring. For example, a purine is always two rings fused
together (6-membered and 5-membered). Pyrimidine is one where it is multisyllable.
b. The pyrimidine ring has three different derivatives: cytosine, uracil (mostly found in RNA), and thymine.
c. Adenine and guanine are purines.
d. Two rings- purine
e. Pyrimidine – one ring
IV. Nucleotide: purines and pyrimidines… [S4]
a. The molecules that were shown before were are all nucleosides.
b. Once you attach a sugar with a phosphate molecule, it becomes a nucleotide.
c. Nucleosides and nucleotides are very different, and you should know the difference.
d. This slide summarizes all of the nucleotides one would commonly encounter in biological systems.
V. Two Types of Pathways Lead to Nucleotides [S5]
a. Synthesis of nucleotides
b. There are two pathways in which the purine ring system is formed.
c. One is the de novo synthesis (in Latin it means “new” or “from scratch”) but in reality, it is not from scratch.
The enzymes are very smart, and in the process of evolution, we have acquired a system where we can take
things from here and there and then fuse them all together and build these purine ring systems.
d. The other pathway is the Salvage pathway. Any food that you eat contains nucleotides and nucleosides. So
there are pathways where we can salvage those. That’s one way of looking at it.
e. Most of what we ingest is not always used in the salvage pathway. It’s a very limited amount. But then the
ribose sugars that are present give us some energy, so that is used in the energy pathway.
f. Most of the time the salvage pathway exists because cells themselves know how to get to these synthesized
nucleotides. And then they recycle them. That is the salvage pathway.
VI. De Novo Purine Nucleotide Synthesis [S6]
a. The starting base material is always a sugar ring in purine synthesis. This is the main difference (between
purines and pyrimidines) you have to note. What is the starting material for purine and pyrimidine synthesis?
Must know this for the exam.
b. In this case, it starts with a ribose sugar molecule.
c. Then, there are about 11 steps that lead to the production of a particular product, which is called inosine
monophosphate.
d. From here, one can make whatever purine you want. Either an Adenosine monophosphate or a guanosine
monophosphate.
e. In this case, the production of nucleotides and nucleosides is like an assembly line. (Analogy to a Honda car
plant where you have a frame and build on top of it. So the base is the same for all cars, but on top there are
changes.) The base of this particular system ends up in the IMP.
f. All of these pathways are very well-controlled in the sense that these enzymes know how to stop themselves
when there is an overproduction of the product. Example is that if you have a car assembly line, and the cars
are not being sold, then you will stop the production line.
g. All of these enzymes have allosteric sites, and they are able to stop themselves from production of product.
VII. John Buchanan [S7]
a. John Buchanan did some research 50 to 60 years ago with pigeons.
b. He noticed that most of the excretions from dove poop contained uric acid.
c. He had a smart idea of labeling the atoms that he was feeding the pigeons with and the analyzed where
these atoms are getting out in the uric acid That is how he realized how aspartic acid, glycine, glutamine,
formate, as well as carbon dioxide play a big role in the formation of the purine rings.
d. This is actually not de novo, but it collects a bunch of things and puts them together.
VIII. De novo Purine Nucleotide Synthesis [S8]
a. There are three amino acids that play a role in the formation of the purine ring, as well as two formates and
one carbon dioxide that is taken out from the respiratory carbon dioxide. This is the source of how the purine
ring is formed.
FUNDAMENTALS HOUR 2
AUGUST 31, 2010
DEIVANAYAGAM
SYNTHESIS AND DEGRADATION OF NUCLEOTIDES
Scribe: FARAH BUTT
Proof: CALVIN SIMS
Page 2 of 5
IX. 11 Steps [S9]
a. There are 11 steps that make up the formation of IMP.
b. It goes down the first column and then comes back up to the top of the second column, and goes back down.
c. The crystal structures of every one of these enzymes involved in the process have been solved.
d. Most people now know how these enzymes work, and in a number of places people have designed
inhibitors.
X. The names of these… [S10]
a. Most important slide.
b. Know committed steps, feedback steps, inhibitors, and diseases
XI. De novo Purine Nucleotide Synthesis [S11]
a. The very first step starts with ribose-5-phosphate.
b. In the first reaction with ATP, it donates two phosphate molecules to the C1 atom and two phosphate atoms
get formed (on PRPP). This product is called 5-phosphororibosyl-1-pyrophosphate (PRPP).
c. This is also the limiting step. It’s also called α- PRPP.
d. If the production of this particular product is not made, then the rest of synthesis does not take place.
XII. Acquisition of Purine Atom [S12]
a. This next step is always called the committed step.
i. A committed step in any enzymatic reaction or pathway is the first step which cannot be reversed. Most of
the others can be reversed by other enzymes, but this step is one that cannot be reversed.
b. This is the first committed step in which the glutamine donates the first purine atom, which is N9.
c. The ring system is numbered slightly differently (take note of it).
d. So first we acquire the N9 atom. The enzyme that is involved is a glutamine PRPP amidotransferase. So it
takes an amide group and it transfers, and that’s why it’s called an amidotransferase.
XIII. Glutamine PRPP Amidotransferase [S13]
a. This particular enzyme in the first step committed step in this process is also one that is subject to feedback
inhibition.
b. You should know this slide very well.
c. This enzyme limits synthesis by feedback inhibition.
d. If there is too much of GDP or ADP being formed, then this enzyme has an allosteric site present in it that is
able to control the reaction.
e. There is also a site that is inhibited by a compound called azaserine. Its molecular structure looks very similar
to glutamine. This enzyme utilizes glutamine and takes the N9 atom.
f. Azaserine binds to it irreversibly.
XIV.
Allosteric Enzyme Cartoon Representation [S14]
a. An allosteric enzyme has a substrate-binding site.
b. When talking about enzymes and substrates before, he left out this group of enzymes because he was
teaching us Michaelis-Menten kinetics. It does not apply to allosteric enzymes because for allosteric
enzymes, when product is overproduced, it will stop itself. So the growth curve he showed us before does
not apply to any of these enzymes. These enzymes will always have a sigmoidal curve.
c. If the allosteric activator (in this case GTP or GDP) doesn’t bind to the site, the substrate will not be able to
bind. So once it goes and locks it and changes the conformation of the protein and allows substrate to bind,
the reaction will take place.
d. On the other hand, there are inhibitors. In our case, there’s too much of purines that are overproduced. The
purines that are overproduced have a similar structure to the activator. So they will come and bind to that
site, and the conformation changes slightly differently. At that point, it will stop the substrate from binding. It
is a highly controlled process.
e. When there is too much something being made, it could become toxic. So these enzymes beautifully control
how much of the product is being made.
f. This particular structure is very important and one of the steps where inhibition could take place.
XV. Acquisition of Purine Atoms [S15]
a. Third step is the only step where three amino acids come together very quickly.
b. It comes from the amino acid glycine.
c. The enzyme is called glycinamide ribonucleotide (GAR) synthetase. And that utilizes ATP and gives a
phosphate molecule as well as adds all these three amino acids to the chain.
d. The fourth step is where the C8 gets added on.
XVI.
Acquisition of Purine Atom N3 [S16]
a. You can see that the structure is close to complete now.
b. Remember this is a five-membered ring. Five-membered rings have to be formed first in the purines.
FUNDAMENTALS HOUR 2
Scribe: FARAH BUTT
AUGUST 31, 2010
Proof: CALVIN SIMS
DEIVANAYAGAM
SYNTHESIS AND DEGRADATION OF NUCLEOTIDES
Page 3 of 5
c. We used a glutamine two steps earlier. The glutamine contributes to the formation of the first FGAM.
d. At this stage, the next enzyme comes into play, which is AIR synthetase, and makes this cyclic ring.
e. This is the imidazole ring that is first formed. There are two nitrogens on it.
f. This is the first five-membered ring that is synthesized in this cycle.
XVII. Acquisition of Purine Atom C6 [S17]
XVIII. Elimination of Fumarate [S18]
a. After the acquisition of the C6 and N1 atoms come into play, there is a big fumarate that is present.
b. Fumarate gets removed by another enzyme in the next step called SAICAR lyase.
c. After that, it acquires one more atom, the C2 atom.
d. Then cyclization takes place with IMP synthase.
e. We are now at a point where we have come full cycle where we have made a five-membered ring and
synthesized a six-membered ring.
f. If you look at how it’s being made, it starts with the 9th atom and then it keeps on adding. The last one added
is C2.
XIX.
9-457-8-3-6-1~2 [S19]
a. These are the nine steps shown.
b. You will quickly notice in this slide that there are at least two places where folates are being replaced.
c. In these synthesis cycles, folate analogs can be very well used to inhibit these enzymes.
d. In most of these steps, you’ll see that ATP is applied. So a lot of energy is required and a lot of transfer of
phosphates that are released and taken in.
XX. In vertebrates these reactions are coupled together [S20]
a. In lower life forms, you’ll find that each one of these is present as a separate enzyme. They all have separate
enzymes that carry out this particular work.
b. If you come to vertebrates, you find that many of these enzymes are combined together into one enzyme. In
vertebrates, it is done a little more efficiently.
c. The advantage is that some of the products formed here are intermediates. If you let them out into solution,
most of the time they could react with whatever is present and then they could form something else. Some
hydrolysis or reduction could take place. And then that particular product becomes wasteful production.
d. In higher organisms, some of these 3 or 4 steps are combined together.
e. Picture is missing from powerpoint.
XXI.
Folate Analogs as Antimicrobial and Anticancer Agents [S21]
a. There are two steps in which folate analogs are used.
b. If someone gets sick, there are folate analogs that are used to treat that. It targets the purine synthesis cycle.
c. People also apply this in treating cancer. Cancer cells are rapidly dividing, and that means there is a lot of
protection of nucleic acids. When cells are dividing, they have to become two different moieties and still retain
their original sense.
d. Methotrexate is a highly used drug in treating breast cancer and other cancers.
e. These folate analogs are very good agents in antimicrobrial and anticancer treatments.
XXII. Step 12: Synthesis of Adenine and Guanine Nucleotides [S22]
a. Now we have the main molecule IMP.
b. From here, one can generate anything, whether you want adenosine or GMP. Either one can be produced
from this. Each of these has two steps through which this goes.
c. The first step utilizes an aspartate and it becomes adenylosuccinate. From there, another other enzyme that
is a lyase will cleave the fumarate group and you get AMP production.
d. Similarly, with the formation of GMP, there are two steps. You can look it up yourself. Wants you to notice
that the first steps for both can be inhibited.
e. These steps can be inhibited.
XXIII. The Purine Biosynthetic Pathway is Regulated at Many Steps [S23]
a. The entire pathway has its own way of inhibiting itself when there is overproduction of something and stops
the cycle.
b. All of these products (AMP, ADP, ATP) are able to bind.
c. This enzyme has particular sites for the guanine series, called the G series.
d. For the adenine, there is A series.
e. When the production of one becomes more, it stops making any more of the product it is supposed to make.
f. The second place where the most important inhibition takes place is where IMP is converted to ADP or GDP.
g. When the production of GDP is much higher, it stops at the step where it is converting. There is no point at
stopping at the third or fourth step. All of these enzymes are very smartly evolved. They have acquired the
ability to understand the production mechanism and inhibit making more of what is not necessary.
FUNDAMENTALS HOUR 2
Scribe: FARAH BUTT
AUGUST 31, 2010
Proof: CALVIN SIMS
DEIVANAYAGAM
SYNTHESIS AND DEGRADATION OF NUCLEOTIDES
Page 4 of 5
h. Mostly in purine synthesis, there are two sites (for regulation): Gln PRPP and IMP
dehydrogenase/adenylosuccinate synthetase. Highlight this in your notes.
i. Know this slide. This will be one of the questions you should know. Where it can be regulated.
XXIV. Can Cells Salvage Purines? [S24]
a. First pathway was de novo pathway, and we looked at that.
b. Second pathway is the salvage pathway.
c. Can we salvage purines? Yes, you can salvage purines.
d. There are specific nucleotidases. They take any monophosphate, and through hydrolysis, it takes the
phosphate off and releases the nucleoside.
e. If it has the ring structure as well as the base (purine or pyrimidine), it’s always called a nucleoside. Once
the phosphate group gets attached to the sugar rings, it becomes a nucleotide.
f. These nucleosides have other enzymes that can work on it, which can separate the base from the ribose ring.
g. Most of these times these bases are utilized so that they are fused back onto a ribose ring and then that
becomes the salvage pathway.
h. In our system most of the time, particularly when we’re eating, we ingest a lot of nucleotides. But most of
them get excreted out.
i. The fusion of the base to the ribose ring takes place in a reaction which involves HGPRT, which is an
important reaction. There are diseases associated with this particular reaction.
XXV. Major Pathways of Purine Catabolism in Animals [S25]
a. The nucleoside is usually degraded to a inosine, xanthosine, or guanosine.
b. All of these nucleosides are converted to an intermediate step.
c. At this point, the PNP enzyme (which was thought to be a drug target in cancer therapy, but only involved in
salvage pathway and not in synthesis pathway. It is much better to block synthesis pathway in cancer
treatment) further converts each one of these molecules (inosine to hypoxanthine, xanthonine to xanthine,
and guanosine to guanine).
d. All of these three then get converted into uric acid by the xanthine oxidase.
e. Most of the time we secrete this uric acid in our excretions.
f. All mammals and all vertebrates do this, so it is a common pathway.
XXVI. Major Pathways of Purine Catabolism in Animals [S26]
XXVII. Animals Oxidize Uric Acid [S27]
a. Some animals further break it down into products that are either somewhat useful to them or not useful to
them.
b. Bird secrete as uric acid.
c. Others secrete them as allanoins.
d. Fish and amphibians convert it to urea.
e. Other invertebrates use urease to convert urea to simple ammonia molecules.
f. So the excretion of these compounds is different in different life forms.
XXVIII. HGPRT Converts Bases Back to Nucleotides [S28]
a. HGPRT converts bases back to nucletodies.
b. α- PRPP was the first limiting step. Without this product, none of these reactions would happen. If this is
available then hypoxanthine can combine to this enzyme HGPRT and form IMP.
c. α-PRPP is a critical product. It can go into de novo synthesis as well as be utilized in the salvage pathway.
Neither de novo nor salvage pathway would work without PRPP.
XXIX. Lesch-Nyhan Syndrome [S29]
a. Lesch-Nyhan syndrome is one of the diseases in which HGPRT is absent.
b. It leads to a severe disorder.
c. Results in accumulation of uric acid and swelling in joints (arthritis).
d. This enzyme (HGPRT) is shown to be the main cause of this syndrome.
XXX. Major Pathways of Purine Catabolism in Animals [S30]
XXXI. Lack of Adenosine Deaminase [S31]
a. Adenosine deaminase is one of the enzymes that could lead to another set of diseases. And although this is
not shown to be the only one, it has been shown that about 30% of those with severe combined
immunodeficiency syndrome lack this enzyme.
b. The process gets stopped in the salvage pathway.
c. There is an unnecessary accumulation and other intermediates in the breakdown of the nucleic acids.
XXXII. Gout is a Disease [S32]
a. Gout is another disease where people have excess uric acid present in their excretions.
b. Kidney stone is an excess amount of uric acid that has not been thrown out.
c. The enzyme involved is xanthine oxidase. You can read how it plays a big role.
FUNDAMENTALS HOUR 2
Scribe: FARAH BUTT
AUGUST 31, 2010
Proof: CALVIN SIMS
DEIVANAYAGAM
SYNTHESIS AND DEGRADATION OF NUCLEOTIDES
Page 5 of 5
d. Allopurinol is one of the drug inhibitors of xanthine oxidase used to treat these diseases.
XXXIII. Summary of Disorders of Purine Metabolism
a. Summary of all disorders that can come in the absence of any of these enzymes from the pathway.
b. Doesn’t expect us to know all 11 steps of the cycle by name. At least know why folate analogs can be used
to stop synthesis. And, know how these enzymes (allosteric enzymes) control themselves very well and
inhibit themselves.
[End 40:08 mins]