Chapter 23: Nucleotide Metabolism

Question 1

In this chapter, we consider the nature of the nucleotide biosynthetic pathways. In doing so, we will examine how they are regulated and the consequences of their blockade, both by genetic defects and through the administration of chemotherapeutic agents. We then discuss how nucleotides are degraded. In following the general chemical themes of nucleotide metabolism, we will break our discussion into sections on purines, pyrimidines, and deoxynucleotides (including thymidylate). The structures and nomenclature of the major purines and pyrimidines are given in Table 3-1

Question 2

What are purine nucleotides initially derived from?

Answer 2

Purines are initially formed as ribonucleotides rather than as free bases.
The ribonucleotides form IMP which then synthesize AMP and GTP and finally

purine biosynthesis steps:
Ribose-5-Phosphate to IMP synthesis
Synthesis of AMP from IMP
Synthesis of GMP from IMP

Question 3

IMP is the precursor of both?

Answer 3

Inosine Monophosphate yields Adenine and Guanine ribonucleotides, AMP and GMP

Question 4

Do we need to know the 11 reactions involved in the synthesis of IMP, AMP, or GMP?

Answer 4

pg.807

Question 5

How does AMP differ from IMP?

Answer 5

AMP, which differs from IMP only in the replacement of its 6-keto group by an amino group, is synthesized in a two-reaction pathway

Question 6

How are Nucleoside Diphosphates and Triphosphates (ATP and GTP) Synthesized ?

Answer 6

By the Phosphorylation of Nucleoside Monophosphates such as AMP and GMP. This is done by Kinases.

To participate in nucleic acid synthesis, nucleoside monophosphates must first be converted to the corresponding nucleoside triphosphates.
First, nucleoside diphosphates are synthesized from the corresponding nucleoside monophosphates by base-specific nucleoside monophosphate kinases.
-For example, adenylate kinase catalyzes the phosphorylation of AMP to ADP: AMP + ATP⇌2 ADP
-Similarly, GDP is produced by guanylate kinase: GMP + ATP⇌ GDP + ADP
(These nucleoside monophosphate kinases do not discriminate between ribose and deoxyribose in the substrate.)
Nucleoside diphosphates are converted to the corresponding triphosphates by nucleoside diphosphate kinase; for instance,
GDP + ATP ⇌ GTP + ADP

In other words, Kinases convert IMP-derived AMP and GMP to ATP and GTP.

Question 7

How is Purine nucleotide synthesis regulated?

Answer 7

Purine nucleotide synthesis is regulated by feedback inhibition and feedforward activation.

amidophosphoribosyl transferase: different inhibitor binding sites for adenine and guanine nucleotides

The rate of synthesis of GMP increases with [ATP], whereas that of AMP increases with [GTP].

The pathways synthesizing IMP, ATP, and GTP are individually regulated in most cells so as to control the total amounts of purine nucleotides available for nucleic acid synthesis, as well as the relative amounts of ATP and GTP.

The rate of IMP production is therefore independently but synergistically controlled by the levels of adenine nucleotides and guanine nucleotides.

Question 8

In most cells, the turnover of nucleic acids, particularly some types of RNA, causes what to release? What happens to them?

Answer 8

Releases adenine, guanine, and hypoxanthine. These free purines are reconverted to their corresponding nucleotides (AMP, IMP, GMP) through salvage pathways.
Purine Salvage is a biochemical pathway that recycles partially degraded purine bases to reform purine nucleotides./Salvage reactions convert free purine and pyrimidine bases into nucleotides.

Question 9

What occurs in the salvage pathways?

Answer 9

In contrast to the de novo purine nucleotide synthetic pathway, which is virtually identical in all cells, salvage pathways are diverse in character and distribution. In mammals, purines are mostly salvaged by two different enzymes. Adenine phosphoribosyltransferase (APRT) mediates AMP formation using PRPP:
Adenine + PRPP ⇌ AMP + PPi

Hypoxanthine–guanine phosphoribosyltransferase (HGPRT) catalyzes the analogous reaction for both hypoxanthine and guanine:

Hypoxanthine + PRPP ⇌ IMP + PPi
Guanine + PRPP ⇌ GMP + PPi

Question 10

Summary of Synthesis of Purine Ribonucleotides

Answer 10

• The purine nucleotide IMP is synthesized in 11 steps from ribose5-phosphate, aspartate, fumarate, glutamine, glycine, and HCO3 − . Purine nucleotide synthesis is regulated at its first and second steps.
• IMP is the precursor of AMP and GMP, which are phosphorylated to produce the corresponding di- and triphosphates.

Question 11

What are pyrimidine ribonucleotides, UTP and CTP derived from?

Answer 11

UMP is the precursor

Question 12

How is UMP synthesized?

Answer 12

UMP Is Synthesized in Six Steps
1. Synthesis of Carbamoyl Phosphate
2. Synthesis of Carbamoyl Aspartate
3. Ring Closure to form dihydroorotate
4. Oxidation of Dihydro Orotate
5. Addition of ribose Phosphate moiety
6. Decarboxylation to form UMP

UMP is synthesized as a pyrimidine base to which ribose-5-phosphate is added.

Question 13

Difference between purine and pyrimidine nucleotide synthesis regarding the ring.

Answer 13

In contrast to purine nucleotide synthesis, the pyrimidine ring is coupled to the ribose-5-phosphate moiety after the ring has been synthesized.

Question 14

How is UMP converted to UTP and CMP?

Answer 14

The synthesis of UTP from UMP is analogous to the synthesis of purine nucleoside triphosphates (Section 23-1B). The process occurs by the sequential actions of a nucleoside monophosphate kinase and nucleoside diphosphate kinase:

1. UMP + ATP ⇌ UDP + ADP
2. UDP + ATP ⇌ UTP + ADP

CTP is formed by the amination of UTP by CTP synthetase (Fig. 23-7). In animals, the amino group is donated by glutamine, whereas in bacteria it is supplied directly by ammonia.

Question 15

Where is Pyrimidine Nucleotide Biosynthesis regulated?

Answer 15

Pyrimidine Nucleotide Biosynthesis Is Regulated at ATCase or Carbamoyl Phosphate Synthetase II

In bacteria, the pyrimidine biosynthetic pathway is primarily regulated at Reaction 2, the ATCase reaction (Fig. 23-8a). In E. coli, control is exerted through the allosteric stimulation of ATCase by ATP and its inhibition by CTP (Section 12-3). In many bacteria, however, UTP is the major ATCase inhibitor.

Question 16

Summary of Synthesis of Pyrimidine Ribonucleotides

Answer 16

• The pyrimidine nucleotide UMP is synthesized from 5-phosphoribosyl pyrophosphate, aspartate, glutamine, and HCO 3 − in six reactions. UMP is converted to UTP and CTP by phosphorylation and amination.
• Pyrimidine nucleotide synthesis is regulated in bacteria at the ATCase step and in animals at the step catalyzed by carbamoyl phosphate synthetase II.

Question 17

How does DNA chemically differ from RNA?

Answer 17

DNA differs chemically from RNA in two major respects: (1) Its nucleotides contain 2′-deoxyribose residues rather than ribose residues, and (2) it contains the base thymine (5-methyluracil) rather than uracil. In this section, we consider the biosynthesis of these DNA components.

Question 18

How are deoxyribonucleotides synthesized?

Answer 18

Deoxyribonucleotides are synthesized from their corresponding ribonucleotides by the reduction of their C2′ position rather than by their de novo synthesis from deoxyribose-containing precursors.

Ribonucleotide Reductase uses a free radical mechanism to Convert Ribonucleotides to Deoxyribonucleotides

uses NDP as substrate

Ribonucleotide Reductase Converts NDPs to dNDPs

Question 19

RNRs

Answer 19

(Enzymes that catalyze the formation of deoxyribonucleotides by the reduction of the corresponding ribonucleotides are named ribonucleotide reductases (RNRs). There are three classes of RNRs, which differ in their prosthetic groups, although they all replace the 2′-OH group of ribose with H via a free-radical mechanism involving a thiyl radical)

Here we discuss the mechanism of Class Ia RNRs, which have an Fe-containing prosthetic group and which occur in all eukaryotes and many aerobic bacteria (Class Ib RNRs have a similar mechanism but have an Mn-containing prosthetic group)

Class Ia RNRs reduce ribonucleoside diphosphates (NDPs) to the corresponding deoxyribonucleoside diphosphates (dNDPs). T

Question 20

Do we know the detailed steps of the Ribonucleotide Reductase mechanism like Thioredoxin?

Answer 20

pg. 815

1.3′-Ribonucleotide 1 radical formation
2.Base-catalyzed elimination of water and radical migration
3.Disulfide bond 3 formation
4.Re-formation of 4 the thiyl radical
5.Reduction of the 5 disulfide bond

Question 21

What reduces Ribonucleotide Reductase ?

Answer 21

• newly formed disulfide bond has to be re-reduced by electron (hydrogen) donor, Thioredoxin

Question 22

regulation of ribonucleotide reductase ?

Answer 22

activity site: ATP binding activates enzyme, dATP binding inhibits

a4 b 4 form, inactive, induced by dATP binding to activity site

Question 23

The final step in the production of all dNTPs is

Answer 23

the phosphorylation of the corresponding dNDPs:
dNDP + ATP⇌dNTP + ADP
(ATP can be any NTP or dNTP)

This reaction is catalyzed by nucleoside diphosphate kinase, the same enzyme that phosphorylates NDPs (Section 23-1B). As before, the reaction is written with ATP as the phosphoryl donor, although any NTP or dNTP can function in this capacity.

Question 24

How is Thymine (dTTP) synthesized?

Answer 24

The dTTP substrate for DNA synthesis is derived from dUTP, which is hydrolyzed to dUMP by dUTP diphosphohydrolase (dUTPase):
dUTP + H2O → dUMP + PPi

The dUMP is then methylated to generate dTMP by thymidylate synthase:
dUMP + N5 ,N10 -methylene-THF → dTMP + dihydrofolate

Finally, the dTMP is phosphorylated to form dTTP by nucleoside monophosphate kinase and nucleoside diphosphate kinase:
dTMP → dTDP → dTTP

Question 25

What is the reason for the energetically wasteful process of dephosphorylating dUTP and rephosphorylating dTMP?

Answer 25

is that cells must minimize their concentration of dUTP in order to prevent incorporation of uracil into their DNA (the enzyme system that synthesizes DNA from dNTPs does not efficiently discriminate between dUTP and dTTP; Section 25-2A).

• appears to waste energy
• necessary to keep dUTP concentration low
• DNA polymerase does not discriminate efficiently between dUTP and dTTP

Question 26

In what way is the thymidylate synthase reaction biochemically unique?

Answer 26

The thymidylate synthase reaction is biochemically unique in that it oxidizes THF to DHF because it goes from a methylene to methyl group. No other enzymatic reaction employing a THF cofactor alters this coenzyme’s net oxidation state. The DHF product of the thymidylate synthase reaction has to be returned to its N5,N10-methylene-THF form through two sequential reactions:
(only known C 1 transfer reaction involving THF where the oxidation state of THF is changed)

-THF is oxidzed to DHF; no other reaction alters the THF oxidation state

Question 27

How is THF regenerated from DHF?

Answer 27

Two ways:

1.DHF is reduced to THF by NADPH as catalyzed by dihydrofolate reductase (DHFR;)/THF is regenerated from DHF by the NADPH-dependent activity of dihydrofolate reductase (DHFR)
2.Serine hydroxymethyltransferase (Section 21-4A) transfers the hydroxymethyl group of serine to THF yielding N5 ,N10 -methylene-THF and glycine.

Question 28

Inhibition of thymidylate synthase or DHFR blocks what? What is this the basis for?

Answer 28

Answer: blocks dTMP synthesis and is therefore the basis of certain cancer chemotherapies (inhibitors used in cancer therapy)

Explanation:
-dTMP synthesis is a critical process for rapidly proliferating cells, such as cancer cells, which require a steady supply of dTMP for DNA synthesis. Interruption of dTMP synthesis can therefore kill those cells.

-5-Fluorodeoxyuridylate (FdUMP)is an irreversible inhibitor of thymidylate synthase. This substance, like dUMP, binds to the enzyme (an F atom is not much larger than an H atom) and undergoes the first two steps of the normal enzymatic reaction (Fig. 23-15). In Step 3, however, the enzyme cannot abstract the F atom as F + (F is the most electronegative element) so that the enzyme is frozen in an enzyme–FdUMP–THF ternary covalent complex.

Question 29

Enzyme inhibitors, such as FdUMP, that inactivate an enzyme only after undergoing part or all of the normal catalytic reaction are called?

Answer 29

Enzyme inhibitors, such as FdUMP, that inactivate an enzyme only after undergoing part or all of the normal catalytic reaction are called mechanism-based inhibitors (alternatively, suicide substrates because they cause the enzyme to “commit suicide”). Because of their extremely high specificity, mechanism-based inhibitors are among the most useful therapeutic agents.

Question 30

aminopterin, amethopterin and trimethoprim do what?

Answer 30

inhibit dihydrofolate reductase (DHFR), thus blocking THF-dependent reactions because the THF converted to DHF by the thymidylate synthase reaction cannot be regenerated.

Question 31

Thymidylate synthase is inhibited by? Dihydrofolate reductase is inhibited by ?

Answer 31

Thymidylate-FdUMP
Dihydrofolate reductase-the antifolates methotrexate (amethopterin), aminopterin, and trimethoprim

Question 32

Summary of the Formation of Deoxyribonucleotides

Answer 32

• Deoxyribonucleoside diphosphates (dNDPs) are synthesized from the corresponding NDPs in a free radical-mediated oxidation reaction catalyzed by ribonucleotide reductase, which contains a binuclear Fe(III) prosthetic group, a tyrosyl radical, and three redox-active sulfhydrylgroups. Enzyme activity is regenerated through disulfide interchange with thioredoxin.
• Ribonucleotide reductase is regulated by allosteric effectors, which ensure that deoxynucleotides are synthesized in the amounts and ratios required for DNA synthesis.
• dTMP is synthesized from dUMP by thymidylate synthase. The dihydrofolate produced in the reaction is converted back to tetrahydrofolate by dihydrofolate reductase (DHFR).

Question 33

Look at google docs for image representing summary of nucleotide metabolism or pg 822

Answer 33

Nucleotides are synthesized from amino acids and ribose-5-phosphate by pathways in which the base is built onto the sugar (purine synthesis) or the sugar is added to the base (pyrimidine synthesis). Nucleotide catabolism yields a derivative of the base and releases the sugar as ribose-1-phosphate.

Question 34

Most foodstuffs, being of cellular origin, contain nucleic acids. How do dietary nucleic acids survive the acidic medium of the stomach? What happens to the ionic nucleotides that cannot pass through cell membranes?
In other words, what are the 2 fates of nucleotides regarding degradation?

Answer 34

1. they are degraded to their component nucleotides, mainly in the intestine, by pancreatic nucleases and intestinal phosphodiesterases.
2. They are then hydrolyzed to nucleosides by a variety of group-specific nucleotidases and nonspecific phosphatases.

Essentially, there are 2 fates for nucleotide degradation,
1. Salvaged for recycling into new nucleic acids (most cells; from internal, not ingested, nucleic acids).
2. Oxidized (primarily in the intestine and liver) by first converting to nucleosides, then to
–Uric Acid (purines)
–Acetyl-CoA & Oxidation succinyl-CoA

AKA Purine and pyrimidine nucleosides can either be degraded to waste products and excreted or can be salvaged as nucleotide components/Purine and pyrimidine nucleosides can either be degraded to waste products and excreted or salvaged to form NUCLEOTIDE COMPONENTS

Question 35

Out of the 2 fates of nucleic degradation, which is more likely to occur?

Answer 35

Only a small fraction of the bases of ingested nucleic acids are incorporated into tissue nucleic acids. Evidently, the de novo pathways of nucleotide biosynthesis largely satisfy an organism’s need for nucleotides. Consequently, ingested bases are mostly degraded and excreted.

Cellular nucleic acids are also subject to degradation as part of the continual turnover of nearly all cellular components.

Question 36

nucleosidase
Nucleoside + H2 O ― ⟶ base + ribose

nucleoside phosphorylase
Nucleoside + P i ――― ⟶ base + ribose-1-P

What events do these reactions describe?

Answer 36

Nucleosides may be directly absorbed by the intestinal mucosa or further degraded to free bases and ribose or ribose-1-phosphate through the action of nucleosidases and nucleoside phosphorylases:

Question 37

What are purines broken down into?

Answer 37

Uric Acid

Question 38

ribose-1-phosphate, a product of the reaction catalyzed by purine nucleoside phosphorylase (PNP), is a precursor of PRPP.

Question 39

Adenosine and deoxyadenosine are not degraded by mammalian PNP. Rather, adenine nucleosides and nucleotides are deaminated by?

Answer 39

adenosine deaminase (ADA) and AMP deaminase to their corresponding inosine derivatives, which can then be further degraded.

The nucleoside, adenosine, is then deaminated and hydrolyzed to form hypoxanthine via adenosine deaminase and nucleosidase respectively. Hypoxanthine is then oxidized to form xanthine and then uric acid through the action of xanthine oxidase.

Question 40

What is The Purine Nucleotide Cycle and what does it generate?

Answer 40

This pathway functions in muscle to prime the citric acid cycle by generating fumarate.

-The deamination of AMP to IMP, when combined with the synthesis of AMP from IMP, has the net effect of deaminating aspartate to yield fumarate.
-Has an important metabolic role in skeletal muscle.
-Muscle replenishes its citric acid cycle intermediates with fumarate generated in the purine nucleotide cycle.

Slide:
* important metabolic role in muscle

• muscle activity requires increase in activity of citric acid cycle
• muscle lacks many enzymes for anaplerotic reactions
• uses fumarate generated in the purine nucleotide cycle

Question 41

Why is the purine nucleotide cycle important in muscle metabolism during a burst of activity?

Answer 41

Increases in muscle activity create a demand for an increase in the TCA cycle, in order to generate more NADH for the production of ATP. However, muscle lacks most of the important enzymes of the major anapleurotic reactions (Anaplerotic reactions are those that form intermediates of a metabolic pathway). Muscle replenishes TCA-cycle intermediates in the form of fumarate generated by the purine nucleotide cycle.

Question 42

Function of Xanthine Oxidase

Answer 42

Xanthine Oxidase Is a Mini-Electron-Transport System. Xanthine oxidase converts hypoxanthine (the base of IMP) to xanthine, and xanthine to uric acid

• product H2O2 converted to H2O and O2 by catalase

Question 43

What are the 3 fates of uric acid after it is produced?

Answer 43

1. In humans and other primates, the final product of purine degradation is uric acid, which is excreted in the urine.
2. Those organisms, which do not excrete urea, also catabolize their excess amino acid nitrogen to uric acid via purine biosynthesis. This complicated system of nitrogen excretion has a straightforward function: It conserves water./• excretion of excess nitrogen as uric acid instead of urea conserves water

3.In all other organisms, uric acid is further processed before excretion.Mammals other than primates oxidize it to their excretory product, allantoin, in a reaction catalyzed by the Cu-containing enzyme urate oxidase. A further degradation product, allantoic acid, is excreted by teleost (bony) fish. Cartilaginous fish and amphibia further degrade allantoic acid to urea prior to excretion. Finally, marine invertebrates decompose urea to NH4 + .

Question 44

Excretion of excess nitrogen as uric acid instead of urea conserves what?

Answer 44

Water

Question 45

What is Gout?

Answer 45

Gout Is Caused by an Excess of Uric Acid.Gout is a disease characterized by elevated levels of uric acid in body fluids.

Its most common manifestation is excruciatingly painful arthritic joint inflammation of sudden onset, most often of the big toe (Fig. 23-24), caused by deposition of nearly insoluble crystals of sodium urate. Sodium urate and/or uric acid may also precipitate in the kidneys and ureters as stones, resulting in renal damage and urinary tract obstruction.

The most prevalent cause of gout is impaired uric acid excretion

Slide:
→ formation of (nearly insoluble) crystals of sodium urate → crystals deposited in joints → gout

Question 46

How is Gout treated?

Answer 46

Gout can be treated by administering the xanthine oxidase inhibitor allopurinol.

Xanthine oxidase hydroxylates allopurinol, as it does hypoxanthine, yielding alloxanthine (at left), which remains tightly bound to the reduced form of the enzyme (xanthine oxidase), thereby inactivating it.

Allopurinol consequently alleviates the symptoms of gout by decreasing the rate of uric acid production while increasing the levels of the more soluble hypoxanthine and xanthine.

Question 47

Pyrimidines are broken down to? Difference between it and purines?

Answer 47

Pyrimidines Are Broken Down to Malonyl-CoA and Methylmalonyl-CoA

Animal cells degrade pyrimidine nucleotides to their component bases.
The reactions, like those of purine nucleotides, occur through dephosphorylation, deamination, and glycosidic bond cleavages. The resulting uracil and thymine are then broken down in the liver through reduction (Fig. 23-25, middle) rather than by oxidation as occurs in purine catabolism.

The end products of pyrimidine catabolism, 𝛃 -alanine and 𝛃 -aminoisobutyrate, are amino acids and are metabolized as such. They are converted, through transamination and activation reactions, to malonyl-CoA and methylmalonyl-CoA.

Question 48

Fates of Malonyl-CoA and methylmalonyl-CoA?

Answer 48

Malonyl-CoA is a precursor of fatty acid synthesis (Fig. 20-26), and methylmalonyl-CoA is converted to the citric acid cycle intermediate succinyl-CoA

Question 49

Summary of Nucleotide Degradation?

Answer 49

• Purine nucleotides are degraded by nucleosidases and purine nucleoside phosphorylase (PNP). Adenine nucleotides are deaminated by adenosine deaminase and AMP deaminase. The synthesis and degradation of AMP in the purine nucleotide cycle yield the citric acid cycle intermediate fumarate in muscles. Xanthine oxidase catalyzes the oxidation of hypoxanthine to xanthine and of xanthine to uric acid.
• In primates, birds, reptiles, and insects, the final product of purine degradation is uric acid, which is excreted. Other organisms degrade urate further.
• Pyrimidines are broken down to intermediates of fatty acid metabolism