Purine and Pyrimidine Metabolism Flashcards Preview

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Flashcards in Purine and Pyrimidine Metabolism Deck (42)
1

Purine- and pyrimidine-containing nucleotides are important because these molecules are:

1. The building blocks that make up the nucleic acids (DNA and RNA) which are critical for cell division and gene transcription/translation.

2. The primary energy carriers in the cell in molecules such as ATP and GTP

3. The foundation for many coenzymes such as CoA, FAD, NAD, NADP

4. Important in intracellular signaling such as cAMP, cGMP

5. Carriers for activated intermediates such as UDP-glucose which is directed to glycogen synthesis and CDP-diacylglycerol which is involved in glycerophospholipid synthesis.

2

Nucleotides and their pathways

a. Thus, it is critical that the cell be able to readily make the nucleotides it needs from basic chemical sources or to recycle nucleotides, and also to degrade excess nucleotides and excrete the resulting metabolic products.

b. Since nucleotides are critical for cell division, pharmacological inhibition of these pathways is a strategy that has been used in medications that have anti-cancer, anti-bacterial and anti-viral activities.

c. As we saw with complex lipids, if a person has an inborn error of metabolism where nucleotide synthesis or degradation pathways are disrupted, toxic metabolites can accumulate and cause diseases such as gout and Severe Combined Immunodeficiency Syndrome (SCID).

3

Purine and Pyrimidine

a. There are 2 main parts to purines and pyrimidines:
i. the base which is a single or double ringed structure that contains N, C, O and H
ii. a sugar that may or may not be phosphorylated.

b. A nucleoside is a base combined with a pentose sugar.
i. If the sugar is phosphorylated, then the molecule is called a nucleotide.

4

Nucleotide and Nucleoside

a. A nucleoside is a base combined with a pentose sugar.

b. If the sugar is phosphorylated, then the molecule is called a nucleotide.

5

Where Purines and Pyrimidines come from

a. Most of the purines and pyrimidines that are present in the body come either through de novo synthesis from other component molecules or through ‘recycling’ of pre-existing bases which are combined with sugar moieties.

b. Dietary purines and pyrimidines typically contribute minimally to total body pools of these molecules.

c. Once a nucleotide is made, it is important to be able to alter/interconvert the state of phosphorylation (GTP + ADP --> GDP + ATP).

d. Additionally, ribonucleotides need to be converted to deoxy-ribonucleotides for use in DNA synthesis

6

These Purine and Pyrimidine compounds need to be degraded and excreted or recycled.

We will therefore now discuss:

1. de novo Purine nucleotide synthesis
2. de novo Pyrimidine nucleotide synthesis
3. Changes in phosphorylation states and conversion of rNDPs to dNDPs
4. Nucleotide degradation
5. Salvage pathways
6. Important diseases of nucleotide metabolism
7. Drugs that target these pathways

7

Nucleotides

a. Nucleotides contain purine and pyrimidine bases.

b. There are five common ones:
i. guanine and adenine (purines)
ii. uracil, thymine, and cytosine (pyrimidines).

c. A, G, C, and T are found in DNA, while A, G, C, and U are found in RNA.

d. Unusual bases are found in places like tRNA and rRNA, but these are primarily post-transcriptional modifications of the 5 common bases.

e. Bases, when covalently linked to ribose sugars and phosphates, become nucleotides.

8

Nucleoside vs Nucleotide

a. A nucleoside consists of a nitrogenous base covalently attached to a sugar (ribose or deoxyribose) but without the phosphate group.

b. A nucleotide consists of a nitrogenous base, a sugar (ribose or deoxyribose) and one to three phosphate groups.

Nucleoside = Sugar + Base
Nucleotide = Sugar + Base + Phosphate

9

How Purines and Pyrimidines are made

a. Purines and pyrimidine bases can be made in de novo synthetic pathways (from other molecule precursors), or recycled in salvage pathways.


b. In general, the body uses the salvage pathways for most of its needs.

10

The de novo pathways introduction

a. When the de novo synthesis pathways of the purine and pyrimidine bases are examined, we find they differ in several ways.

b. This includes:
1. whether the base is made on the ribose sugar or made separately (and then added to the sugar)
2. where the atoms come from
3. the intermediates that are produced,
and
4. how these intermediates are converted to yield the full set of bases.

c. The synthesis of the activated sugar moiety is a key regulated step that you will see in several parts of these pathways.
i. The enzyme that catalyzes this key regulated step is PRPP synthetase.

Rxn:
Ribose-5 phosphate---> 5-Phosphoribosyl-1 Pyrophosphate
PRPP Synthetase uses an MG

11

Purine nucleotide synthesis
(purine de novo synthesis)

Lare Overview

a. The purine metabolic/catabolic pathways contain feedback loops and feed into the salvage pathways.

b. The atoms that make up the purine base come from a variety of sources including several amino acids as well as small molecule sources.

c. The purine base is made by starting with a ribose sugar and then building the base on the sugar, one step at a time. Many enzymes are involved.

d. The key regulated step of purine de novo synthesis is at the start, when PRPP (contains the ribose sugar) and glutamine are used by glutamine phosphoribosyl pyrophosphate amidotransferase to add the first nitrogen to the PRPP.

e. The secondary regulated step is conversion of a ribose 5'-phosphate to PRPP.

f. The pathway involves the addition of several amino acids and CO2 to the growing base as well as tetrahydrofolate and ATP as important elements in the pathway.

g. The first base that is produced by this pathway is inosine mono-phosphate (IMP). IMP is then used to make the GMP and AMP bases by the action of enzymes that act on the IMP. Failure of one of the enzymes involved in AMP synthesis can lead to a form of autism.

h. Feedback loops are a critical mechanism of regulation in purine synthesis. Specifically, IMP, GMP, and AMP inhibit enzymes that act early in the pathway.

12

Purine De Novo Synthesis

1. The purine base is made by starting with a ribose sugar and then building the base on the sugar, one step at a time.
i. Many enzymes are involved.

2. The key regulated step of purine de novo synthesis is at the start, when PRPP (contains the ribose sugar) and glutamine are used by glutamine phosphoribosyl pyrophosphate amidotransferase to add the first nitrogen to the PRPP.

3. The secondary regulated step is conversion of a ribose 5'-phosphate to PRPP.

4. The pathway involves the addition of several amino acids and CO2 to the growing base as well as tetrahydrofolate and ATP as important elements in the pathway.

5 The first base that is produced by this pathway is inosine mono-phosphate (IMP).
i. IMP is then used to make the GMP and AMP bases by the action of enzymes that act on the IMP.
ii. Failure of one of the enzymes involved in AMP synthesis can lead to a form of autism.

*Feedback loops are a critical mechanism of regulation in purine synthesis.
i. Specifically, IMP, GMP, and AMP inhibit enzymes that act early in the pathway.

13

The key regulated step of purine de novo synthesis is..

a. The key regulated step of purine de novo synthesis is at the start, when PRPP (contains the ribose sugar) and glutamine are used by glutamine phosphoribosyl pyrophosphate amidotransferase to add the first nitrogen to the PRPP.

b. Feedback loops are a critical mechanism of regulation in purine synthesis.
i. Specifically, IMP, GMP, and AMP inhibit enzymes that act early in the pathway.

14

What is the end bases of Purine Nucleotide Synthesis

a. The first base that is produced by this pathway is inosine mono-phosphate (IMP).

b. IMP is then used to make the GMP and AMP bases by the action of enzymes that act on the IMP.

c. Both adenine and guanine are derived from the nucleotide inosine monophosphate (IMP), which is the first compound in the pathway to have a completely formed purine ring system.

d. Failure of one of the enzymes involved in AMP synthesis can lead to a form of autism.

15

Purine Biosynthesis
According to Wiki

(Strong and good summary)

a. Purines are biologically synthesized as nucleotides and in particular as ribotides, i.e. bases attached to ribose 5-phosphate
i. The purine base is made by starting with a ribose sugar and then building the base on the sugar, one step at a time.
ii. Many enzymes are involved

b. The first committed step is the reaction of PRPP, glutamine and water to 5'-phosphoribosylamine (PRA), glutamate, and pyrophosphate
i. catalyzed by glutamine phosphoribosyl pyrophosphate amidotransferase , which is activated by PRPP and inhibited by AMP, GMP and IMP.
ii. Need to know this for test!

Many Enzyme Reactions..

c. The last step is catalyzed by Inosine monophosphate synthase.
i. The first base that is produced by this pathway is inosine mono-phosphate (IMP). IMP is then used to make the GMP and AMP bases by the action of enzymes that act on the IMP.
FAICAR → IMP + H2O

d. Both adenine and guanine are derived from the nucleotide inosine monophosphate (IMP), which is the first compound in the pathway to have a completely formed purine ring system.

16

Glutamine phosphoribosyl pyrophosphate amidotransferase

a. The key regulated step of purine de novo synthesis is at the start, when PRPP (contains the ribose sugar) and glutamine are used by glutamine phosphoribosyl pyrophosphate amidotransferase to add the first nitrogen to the PRPP.

b. Wiki-->The first committed step is the reaction of PRPP, glutamine and water to 5'-phosphoribosylamine (PRA), glutamate, and pyrophosphate catalyzed by glutamine phosphoribosyl pyrophosphate amidotransferase

glutamine phosphoribosyl pyrophosphate amidotransferase -->which is activated by PRPP and inhibited by AMP, GMP and IMP.

17

Pyrimidine nucleotide synthesis

Large Summary

a. The atoms that make up the pyrimidine bases come from both amino acid and small molecule sources.

b. Unlike the purines, the pyrimidine base ring is not made on the ribose sugar, but made separately and then the base ring is added to the sugar.

c. The first step in the synthesis of the pyrimidine ring is the primary source of regulation.
i. The enzyme that catalyzes this key step is carbamoyl phosphate synthetase II.
ii. This is different from carbamoyl phosphate synthetase I which you heard about in the urea cycle in that it is in the cytosol, and it is activated by PRPP and inhibited by UTP.

d. The first nucleotide produced by this pathway is uracil mono-phosphate (UMP). To make cytosine, the nucleotides must first be converted to a triphosphate form.
i. Once UMP is converted to UTP (see below), it can be converted to CTP by the action of the CTP synthase enzyme.

18

Pyrimidine nucleotide synthesis

a. The first step in the synthesis of the pyrimidine ring is the primary source of regulation.
i. The enzyme that catalyzes this key step is carbamoyl phosphate synthetase II.
ii. This is different from carbamoyl phosphate synthetase I which you heard about in the urea cycle in that it is in the cytosol
iii. Carbamoyl phosphate synthetase II it is activated by PRPP and inhibited by UTP.

b. The first nucleotide produced by this pathway is uracil mono-phosphate (UMP).

c. To make cytosine, the nucleotides must first be converted to a triphosphate form.
i. Once UMP is converted to UTP (see below), it can be converted to CTP by the action of the CTP synthase enzyme.

19

Carbamoyl phosphate synthase II

*know this enzyme

a. In pyrimidine biosynthesis, it serves as the rate-limiting enzyme and catalyzes the following reaction
i. dont need to know reaction

b. It is activated by ATP and PRPP and it is inhibited by UMP (Uridine monophosphate, the end product of the pyrimidine synthesis pathway).

20

What base is made at the end of Pyrimidine synthesis

a. The first nucleotide produced by this pathway is uracil mono-phosphate (UMP).
i. UMP will inhibit the rate limiting enzyme for pyrimidine synthesis (Carbamoyl phosphate synthase II)

b. To make cytosine, the nucleotides must first be converted to a triphosphate form.

c. Once UMP is converted to UTP (see below), it can be converted to CTP by the action of the CTP synthase enzyme.

21

Changes in phosphorylation states and conversion of rNDPs to dNDPs

Large Overview

a. To convert nucleotide monophosphates (NMPs) to the diphosphate (NDP) and triphosphate (NTP) forms, a set of enzymes called kinases are used.
i. The enzymes take phosphate from an ATP donor and transfer it to other nucleotides.

b. At this point in our lecture, we have covered how the 4 nucleotides needed to make RNA are made.
i. However, in order to make DNA, we need deoxynucleotides.

c. The reduction reaction that converts ribose to deoxyribose is catalyzed by an enzyme called ribonucleotide reductase.
i. Ribonucleotide reductase operates on diphosphates (NDPs; ADP, GDP, CDP, and UDP).

d. Ribonucleotide reductase is regulated by a complex mechanism that ‘senses’ the concentration of dNTPs.
i. Briefly, the enzyme has a primary regulation site (“on/off” switch) that controls the overall activity of the enzyme, and a substrate specificity site (“dial”).
ii. The primary regulation site is active in the presence of ATP, inactive when dATP builds up.
iii. The substrate specificity switch is sensitive to the concentrations of individual dNTPs, and as each builds up, the enzyme changes from operating on one NDP, to operating on another.
iv. This remarkable bit of enzyme regulation ensures that equal and adequate amounts of each NDP are converted to dNDP (and then to the dNTPs).

e. Once UDP is converted to dUDP, it can then be dephosphorylated to make dUMP, which is then converted to dTMP by thymidylate synthase. Kinases can then convert dTMP to dTDP and dTTP.

22

At this point in our lecture, we have covered how the 4 nucleotides needed to make RNA are made.

a. However, in order to make DNA, we need deoxynucleotides.

b. The reduction reaction that converts ribose to deoxyribose is catalyzed by an enzyme called ribonucleotide reductase.

c. Ribonucleotide reductase operates on diphosphates (NDPs; ADP, GDP, CDP, and UDP).

23

Ribonucleotide reductase is regulated by a complex mechanism that ‘senses’ the concentration of dNTPs.

The reduction reaction that converts ribose to deoxyribose is catalyzed by an enzyme called ribonucleotide reductase.

a. Briefly, the enzyme has a primary regulation site (“on/off” switch) that controls the overall activity of the enzyme, and a substrate specificity site (“dial”).

b. The primary regulation site is active in the presence of ATP, inactive when dATP builds up
i. are activated by binding ATP or inactivated by binding dATP to the activity site located on the RNR

c. The substrate specificity switch is sensitive to the concentrations of individual dNTPs, and as each builds up, the enzyme changes from operating on one NDP, to operating on another.

d. This remarkable bit of enzyme regulation ensures that equal and adequate amounts of each NDP are converted to dNDP (and then to the dNTPs).

24

Ribonucleotide reductase (RNR)

a. Ribonucleotide reductase (RNR), also known as ribonucleoside diphosphate reductase, is an enzyme that catalyzes the formation of deoxyribonucleotides from ribonucleotides.
i. Deoxyribonucleotides in turn are used in the synthesis of DNA.

b. Furthermore, RNR plays a critical role in regulating the total rate of DNA synthesis so that DNA to cell mass is maintained at a constant ratio during cell division and DNA repair.
i. are activated by binding ATP or inactivated by binding dATP to the activity site located on the RNR

c. A somewhat unusual feature of the RNR enzyme is that it catalyzes a reaction that proceeds via a free radical mechanism of action.

d. The substrates for RNR are ADP, GDP, CDP and UDP.

25

Regulation of Ribonucleotide reductase (RNR)

a. Regulation of RNR is designed to maintain balanced quantities of dNTPs.

b. Binding of effector molecules either increases or decreases RNR activity.

c. When ATP binds to the allosteric activity site, it activates RNR. In contrast, when dATP binds to this site, it deactivates RNR.

d. In addition to controlling activity, the allosteric mechanism also regulates the substrate specificity and ensures the enzyme produces an equal amount of each dNTP for DNA synthesis

26

Nucleotide degradation

Large Summary

a. Just as nucleotides are made in the body, they must be broken down in order to get rid of excess nucleotides.
i. In fact, we consume far more nucleotides in our diet than we ever need, and so much of these are degraded and excreted by the intestine.

b. Degradation of purine nucleotides occurs by first removing the base from the sugar, yielding a free base (adenosine or guanine).
i. The free bases are then further broken down to uric acid, which is what is excreted from the body in urine.
ii. Failures in this pathway lead to several diseases (see below)

c. Pyrimidines are broken down by first removing the base ring from the ribose, as in purine degradation.
i. However, unlike purine degradation, the base ring is then opened up (the uric acid from purine degradation is a closed ring).
ii. Ultimately, the breaking down of the base ring leads to molecules that can be used in other pathways (Succinyl-CoA, Malonyl-CoA, and Acetyl-CoA).
iii. These products are water soluble and so do not cause problems like uric acid can.

27

Degradation of purine

a. Degradation of purine nucleotides occurs by first removing the base from the sugar, yielding a free base (adenosine or guanine).

b. The free bases are then further broken down to uric acid, which is what is excreted from the body in urine.

c. Failures in this pathway lead to several diseases (see below)

28

Degradation of pyrimidine

a. Pyrimidines are broken down by first removing the base ring from the ribose, as in purine degradation.

b. However, unlike purine degradation, the base ring is then opened up (the uric acid from purine degradation is a closed ring).

c. Ultimately, the breaking down of the base ring leads to molecules that can be used in other pathways (Succinyl-CoA, Malonyl-CoA, and Acetyl-CoA).

d. These products are water soluble and so do not cause problems like uric acid can.

29

Savage Pathway
Introduction

a. Nucleotides can be made de novo from other molecules in the body, but in addition, they can be made through salvage pathways, in which partially degraded nucleotides are reused.

b. The salvage pathways involve enzymes that take free bases and attach them to ribose sugar in the form of PRPP.
i. Failure of these enzymes (transferases) can lead to disease (see below).

30

There are important diseases of nucleotide metabolism

Many diseases are associated with defects in the nucleotide synthesis or degradation pathways. We’ll focus on a few from the purine pathways.

a. Severe Combined immunodeficiency syndrome: caused by a mutation in the gene encoding adenosine deaminase, an enzyme used in the purine degradation pathway.
i. This leads to a buildup of dATP, which inhibits ribonucleotide reductase, which prevents enough dNTPs from being made.
ii. Rapidly proliferating cells (such as those in the immune system) are affected.

b. Gout: caused by a buildup of uric acid in the blood.
i. Uric acid is the result of the purine degradation pathway.
ii. This disease can be caused by deficiencies or hyperactivities of some enzymes, and several risk factors (age, diet, etc.) are associated with the disease.

c. Lesch-Nyhan syndrome: caused by a deficiency in one of the primary enzymes in the purine salvage pathway (HGPRT), leading to higher rates of de novo synthesis of purines.
i. Patients may have gout symptoms, self-mutilating behavior and other severe mental disorders.

31

Severe Combined immunodeficiency syndrome:

a. Severe Combined immunodeficiency syndrome: caused by a mutation in the gene encoding adenosine deaminase, an enzyme used in the purine degradation pathway.

b. This leads to a buildup of dATP, which inhibits ribonucleotide reductase, which prevents enough dNTPs from being made.

c. Rapidly proliferating cells (such as those in the immune system) are affected.

32

Gout:

a. Gout: caused by a buildup of uric acid in the blood.

b. Uric acid is the result of the purine degradation pathway.

c. This disease can be caused by deficiencies or hyperactivities of some enzymes, and several risk factors (age, diet, etc.) are associated with the disease

33

Lesch-Nyhan syndrome:

a. Lesch-Nyhan syndrome: caused by a deficiency in one of the primary enzymes in the purine salvage pathway (HGPRT), leading to higher rates of de novo synthesis of purines.

b. Patients may have gout symptoms, self-mutilating behavior and other severe mental disorders.

34

Drugs can target these pathways

Many drugs have been developed to target the nucleotide synthesis and degradation pathways, or to mimic nucleotides in some way. These include some that are designed to treat gout, but also others that treat cancer or viral infection, for example.

1) Methotrexate and 5-florouracil: targets the thymidylate synthase/folate metabolism cycle (anti cancer)

2) 6-mercaptopurine: inhibits AMP synthesis (anti cancer)

3) Azidothymidine (AZT): inhibits viral polymerase (anti HIV)

4) Cytosine arabinoside (araC): targets DNA polymerase (anti leukeia)

5) Acyclovir (ACV): targets viral DNA polymerase and reverse transcriptase (anti Herpes simplex virus)

6) Acivicin: Gln analog, inhibits nucleotide synthesis (mostly GMP; anti cancer)

35

Key differences in purine and pyrimidine nucleotide de novo synthesis:

Purine:
1. Purine base is made on the ribose
2. Initial nucleotide product is IMP
3. IMP is converted to G and A as a monophosphate

Pyrimidines:
1. Base ring is synthesized then attached to the ribose
2. Initial nucleotide product is UMP
3. UMP is converted to C as a triphosphate


36

PRPP Synthase

(In purine synthesis)

a. Purines are built on a ribose sugar

b. Ribose 5 phosphate comes from HMP shunt

c. First step is allosterically regulated and is important
i. Regulation of purine nucleotide synthesis is through feedback loops
i. end products of AMP and GMP from the IMP conversion will inhibit other portions of the purine pathway
ii. Adenine monophosphate and guanine monophosphate

37

End Steps of Purine Synthesis

*great summary steps

a. Key Step as a reminder is PRPP Synthase
i. Ribose 5 phosphate comes from HMP shunt
ii. First step is allosterically regulated and is important

b. Product at the end of the purine synthesis is nosine monophosphate (IMP)
i. Converting IMP to AMP and GMP (adenine monophosphate and guanine monophosphate)

c. Converting monophosphate forms to di and triphosphate forms

d. Regulation of purine nucleotide synthesis is through feedback loops
i. end products of AMP and GMP from the IMP conversion will inhibit other portions of the purine pathway


38

The Important Key regulated step of pyrimidine synthesis

a. Key Regulated Step: Carbamoyl Phosphate Synthase II

b. Pyrimidine synthesis-->Starts at CO2 and Glutamine
i. Pathway Ends at UMP

c. Sources of Atoms for Pyrimidine Synthesis:
CO2 and Glutamine
Aspartate
Ribose sugar is added last


39

Conversion of ribonucleotides to deoxyribonucleotides

Regulation of Ribonucleotide
Reductase:

a. Activity site regulated by
ATP and dATP: on/off switch
i. ATP is the on portion
ii. dATP is the off

b. Substrate Specificity
Site: determines which
dNTP is made

40

Severe combined immunodeficiency syndrome (SCID)

“Bubble Boy”

a. Patients lack active adenosine deaminase (ADA).

b. ADA can act on adenosine or deoxyadenosine, but if it is missing, deoxyadenosine builds up.

b. Excess deoxyadenosine (dAMP) is converted to dATP, which inhibits ribonucleotide reductase, preventing the synthesis of the other dNTPs.

c. Rapidly proliferating cells are affected, including lymphocytes needed for immune competence.

d. Treated with gene therapy.

41

Gout

a. Associated with elevated uric acid levels in blood (hyperuricemia) and urine.

b. Caused by overproduction of purine nucleotides via the de novo pathway.
i. As these excess purines are degraded, it leads to uric acid.

c. Deposition of uric acid crystals leads to inflammatory response and pain.

d. Leads to long-term cartilage destruction.

42

Lesch-Nyhan syndrome

a. Lesch-Nyhan syndrome (LNS) is a rare, genetic disorder caused by a deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase or HPRT.

b. LNS is characterized by self-mutilating behaviors such as lip and finger biting and/or head banging.

c. Frequently the first symptom is the presence of orange-colored crystal-like deposits (orange sand) in the diapers of affected infants.

d. The deposits, which are called urate crystal formation, are caused by increased levels of uric acid in the urine.
i. Uric acid levels, which are abnormally high in individuals with LNS, may also cause sodium urate crystals to form in the joints, kidneys, central nervous system, and other tissues of the body.

e. Other symptoms of LNS may include kidney stones, blood in the urine, pain and swelling of the joints, difficulty swallowing (dysphagia) and eating, and vomiting, impaired kidney function, irritability, uncontrolled aggressive and/or compulsive actions, muscle weakness (hypotonia), uncontrolled spastic muscle movements, and neurological problems such as involuntary writhing movements of the arms and legs (athetosis) and purposeless repetitive movements (chorea) such as shoulder raising and lowering and/or facial grimacing.

Moderate mental retardation is also common. Some individuals may develop a rare disorder called megaloblastic anemia.