ch.20 (808-824)

Question 1

Describe the reaction that forms PRPP, including the enzyme, cofactors, and reaction details.

Answer 1

1. The enzyme ribose-phosphate diphosphokinase (PRPP synthetase) catalyzes the conversion of ribose-5-phosphate and ATP into PRPP (phosphoribosyl Pyrophosphate) and AMP.
2. This reaction occurs in the cytoplasm and uses one ATP, which is converted to AMP
3. It requires Mg²⁺ or Mn²⁺ as cofactors.
4. The enzyme transfers a pyrophosphate group to the 1’ carbon of ribose-5-phosphate to form PRPP.

Question 2

How is PRPP synthetase regulated, and what activates or inhibits it?

Answer 2

1. PRPP synthetase is activated by inorganic phosphate (Pi), showing a sigmoidal v vs. [Pi] curve, indicating cooperative activation.
2. It is inhibited by several nucleotides, including ADP and GDP (feedback inhibition).
3. ADP competes with ATP, and 2,3-bisphosphoglycerate (2,3-BPG) competes with ribose-5-phosphate.
4. Other nucleotides can also inhibit PRPP synthetase.

Question 3

What are the sources of ribose-5-phosphate for PRPP synthesis?

Answer 3

1. Ribose-5-phosphate is primarily supplied by the pentose phosphate pathway from glucose-6-phosphate.
2. It can also be generated from the phosphorolysis of nucleosides.

Question 4

What pathways and reactions require PRPP?

Answer 4

PRPP is required for:
1) De novo synthesis of purine nucleotides (e.g., PRPP + glutamine → phosphoribosylamine);
2) Salvage of purine bases (PRPP + hypoxanthine/guanine/adenine → IMP/GMP/AMP + PPi);
3) De novo pyrimidine synthesis (PRPP + orotate → OMP + PPi);
4) Pyrimidine base salvage (PRPP + uracil → UMP + PPi);
5) NAD synthesis (PRPP + nicotinate/nicotinamide/quinolate → nicotinate mononucleotide or nicotinamide mononucleotide + PPi).

Question 5

What is the clinical relevance of PRPP synthetase and PRPP levels?

Answer 5

1. Deficiency in PRPP synthetase (e.g., PRS1 mutation) leads to severe clinical outcomes, such as impaired nucleotide synthesis.
2. Excess PRPP can contribute to gout by increasing purine synthesis and uric acid levels.
3. Proper PRPP levels are essential for both de novo synthesis and salvage pathways, and for NAD biosynthesis.

Question 6

Why is glutamine essential in nucleotide biosynthesis, and what are the consequences of its deficiency?

Answer 6

1. Glutamine is a key nitrogen donor in five reactions of de novo nucleotide synthesis.

Question 7

How is de novo nucleotide synthesis regulated in purines and pyrimidines?

Answer 7

1. Purine synthesis is regulated at PRPP amidotransferase, while pyrimidine synthesis is regulated at carbamoyl phosphate synthetase II (CPS II).
2. Regulation of CTP synthetase also ensures the correct cellular ratio of UTP to CTP.
3. These control points help maintain nucleotide balance and synthesis efficiency.

Question 8

Summarize the general features of de novo synthesis of purine nucleotides in mammalian cells.

Answer 8

1. De novo synthesis of purine nucleotides occurs in the cytosol of mammalian cells and uses amino acids as sources of carbon and nitrogen, along with CO₂ and one-carbon units from tetrahydrofolate (H₄-folate).
2. The pathway is energy-intensive, requiring ~6 ATP per IMP molecule.
3. All enzymes are cytosolic, but not all cells (e.g., RBCs) can carry out this synthesis.
4. The pathway produces IMP, which is the precursor to both AMP and GMP.

Question 9

Describe the role of IMP in purine nucleotide synthesis and its regulation.

Answer 9

1. IMP (inosine monophosphate) is the first nucleotide formed in de novo purine synthesis and serves as the common precursor for AMP and GMP.
2. IMP is not usually found under aerobic conditions due to its rapid conversion.
3. The conversion of IMP to AMP requires GTP, and IMP to GMP requires ATP, creating a reciprocal regulation.
4. This helps maintain balance between adenine and guanine nucleotides.
5. AMP and GMP act as feedback inhibitors at their respective branchpoints.

Question 10

Explain the committed step in purine synthesis, the enzyme involved, and how it’s regulated.

Answer 10

1. The committed step is the formation of 5-phosphoribosylamine from PRPP and glutamine, catalyzed by glutamine PRPP amidotransferase.
2. This enzyme is allosterically regulated: it is activated by PRPP and inhibited by the end products IMP, AMP, and GMP.
3. The enzyme exists as a monomer (active) or dimer (inactive);
4. PRPP promotes monomer formation, while IMP/AMP/GMP promote dimerization.
5. There are separate binding sites for oxypurines (IMP, GMP) and aminopurines (AMP), and inhibition is synergistic when both are present.

Question 11

What are the key energy and carbon/nitrogen sources used in purine nucleotide synthesis?

Answer 11

1. C4, C5, and N7 from glycine;
2. N3 and N9 from glutamine;
3. C2 and C8 from C-H4folate;
4. N1 from aspartate;
5. C6 from CO2.
6. Serine, glycine, tryptophan, and histidine can contribute to the folate pool.

Question 12

Discuss the multifunctional proteins involved in purine nucleotide biosynthesis.

Answer 12

1. Several enzymes in the purine synthesis pathway are organized as multifunctional proteins:
   (1) A trifunctional protein includes glycinamide ribonucleotide synthetase (step 2), transformylase (step 3), and aminoimidazole synthetase (step 5);
   (2) A bifunctional protein includes carboxylase (step 6) and succinocarboxamide synthetase (step 7);
   (3) Another bifunctional protein includes transformylase (step 9) and IMP cyclohydrolase (step 10).
   This organization increases efficiency of the pathway.

Question 13

What enzymes are involved in converting IMP into AMP and GMP, and how is this step regulated?

Answer 13

1. Conversion of IMP to AMP is catalyzed by adenylosuccinate synthetase (rate-limiting) and requires GTP. AMP acts as a competitive inhibitor.
2. Conversion of IMP to GMP is catalyzed by IMP dehydrogenase (IMPDH), requires ATP, and is inhibited by GMP.
3. Two isoforms of IMPDH exist: IMPDH-I (constitutive) and IMPDH-II (linked to cell proliferation).
4. These steps ensure balanced nucleotide pools through feedback inhibition and energy-linked regulation.

Question 14

How do PRPP and glutamine availability affect purine nucleotide synthesis?

Answer 14

1. PRPP is a key activator of glutamine PRPP amidotransferase and its levels vary widely (10–100× below its Km), making it a major regulator.
2. Glutamine levels are typically near the enzyme’s Km, so it doesn’t strongly affect activity under normal conditions.
3. However, in cancer therapy with asparaginase (which depletes glutamine), purine synthesis can be inhibited due to limited substrate availability.

Question 15

What is the clinical significance of purine synthesis regulation, and how can it be disrupted?

Answer 15

1. Proper regulation is crucial to prevent overproduction of purines and uric acid.
2. Loss of feedback control (e.g., due to enzyme mutations) can lead to excess purine synthesis and uric acid accumulation, contributing to gout.
3. Methotrexate, a chemotherapy drug, inhibits folate recycling and disrupts tetrahydrofolate availability, impairing purine synthesis and DNA replication, making it cytotoxic.

Question 16

Front

Answer 16

Back

Question 17

What are the two main salvage pathways for purines, and how do they function?

Answer 17

1. There are two salvage pathways for purines: one salvages free purine bases (hypoxanthine, guanine, adenine), and the other salvages purine nucleosides.
2. The base salvage pathway uses phosphoribosyl transferases that transfer a ribose-phosphate group from PRPP.
3. HGPRTase salvages hypoxanthine and guanine to form IMP and GMP respectively, while APRTase salvages adenine to form AMP.
4. These enzymes are substrate-specific and regulated by end-product inhibition: IMP and GMP inhibit HGPRTase; AMP inhibits APRTase.

Question 18

How is adenine generated for the salvage pathway involving APRTase, and what is its significance?

Answer 18

1. Adenine for salvage by APRTase is mainly generated during polyamine synthesis.
2. Spermine formation releases 5’-methylthioadenosine, which is degraded by methylthioadenosine phosphorylase to produce adenine and 5-methylthioribose-1-phosphate.
3. The adenine is salvaged by APRTase to form AMP.
4. This process helps regulate de novo purine synthesis by consuming PRPP and generating AMP.

Question 19

How does purine salvage regulate de novo purine synthesis?

Answer 19

Purine salvage regulates de novo synthesis by two mechanisms:
1) Consuming PRPP, a key substrate and positive effector of the de novo pathway, and
2) Producing AMP, IMP, and GMP, which act as negative feedback inhibitors of PRPP amidotransferase.
3) This dual regulation reduces the formation of 5-phosphoribosylamine and slows de novo synthesis.

Question 20

What are the clinical consequences of HGPRTase and APRTase deficiencies?

Answer 20

1. HGPRTase deficiency leads to Lesch-Nyhan syndrome, which is marked by hyperuricemia, mental retardation, and self-mutilation.
2. APRTase deficiency does not cause neurological symptoms but results in excess urinary excretion of adenine, 8-hydroxyadenine (8-HA), and 2,8-dihydroxyadenine (2,8-DHA).
3. 2,8-DHA can form kidney stones that resemble urate stones.
4. Allopurinol helps reduce 2,8-DHA formation and associated nephrotoxicity.

Question 21

How are purine nucleosides salvaged, and what is the role of adenosine kinase?

Answer 21

1. Purine nucleosides, such as adenosine, are salvaged by nucleoside kinases—mainly adenosine kinase, which is a 5’-phosphotransferase that uses ATP to add a phosphate group to nucleosides.
2. These enzymes are specific to different nucleosides.
3. In cells like erythrocytes, which lack PRPP amidotransferase and cannot make nucleotides from scratch (de novo), this salvage pathway is essential to maintain their nucleotide levels.

Question 22

How are adenine and guanine nucleotide levels balanced through interconversion?

Answer 22

1. Adenine and guanine nucleotides are balanced through indirect interconversion via IMP.
2. GMP is converted to IMP by GMP reductase, which is activated by GTP and inhibited by XMP.
3. AMP is converted to IMP by AMP deaminase, which is activated by K+ and ATP and inhibited by Pi, GDP, and GTP.
4. This allows cells to maintain balanced pools of AMP and GMP depending on need.

Question 23

How is GTP involved in tetrahydrobiopterin synthesis, and why is this important?

Answer 23

1. GTP is the precursor for tetrahydrobiopterin (BH4), synthesized via GTP cyclohydrolase I (rate-limiting), G-pyruvoyl-tetrahydropterin synthase, and sepiapterin reductase.
2. BH4 is a cofactor in hydroxylation reactions involving phenylalanine, tyrosine, and tryptophan, and in nitric oxide synthesis.
3. Inhibitors of IMP dehydrogenase lower GTP and BH4 levels, showing the importance of GTP and its synthetic pathway.

Question 24

Describe the degradation pathway of purine nucleotides, nucleosides, and bases.

Answer 24

1. Purines are degraded to uric acid via several steps.
2. Specific nucleases break down RNA/DNA, nucleotidases remove phosphate groups, and deaminases remove amino groups.
3. Purine nucleoside phosphorylase (PNP) converts inosine, guanosine, and xanthosine into their free bases and ribose-1-phosphate.
4. While PNP reactions are reversible, in vivo conditions favor degradation due to low levels of free bases and ribose-1-P.

Question 25

What are the clinical effects of adenosine deaminase and purine nucleoside phosphorylase deficiencies?

Answer 25

1. Adenosine deaminase (ADA) deficiency results in severe combined immunodeficiency (SCID), due to toxic accumulation of deoxyadenosine metabolites. 2. Purine nucleoside phosphorylase (PNP) deficiency causes defective T-cell function but spares B-cell immunity, leading to a selective immunodeficiency syndrome.