Biochemistry Exam Iii - Purines/pyrimidines/porphyrins

Front 1

- Precursors for the synthesis of DNA and RNA.

- Components of coenzymes, e.g. NAD(H), NADP(H), FMN(H2) and CoA.

- Energy currency, driving many metabolic processes, e.g. ATP and GTP

- Carriers in biosynthesis, e.g., UDP for carbohydrates and and CDP for lipids

- Modulators of allosteric regulation of metabolism

- Second messengers, e.g., cAMP and cGMP

Back 1

Nucleotides.
- molecules composed of a pentose, a nitrogenous base and phosphate.

- Key elements in cell physiology

Front 2

Short name, big base

Back 2

Purine

Front 3

Long name, short base

Back 3

Pyrimidine

Front 4

Synthetic pathway purine

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Hypoxanthine

Front 5

Degradation pathway purine

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Xanthine

Front 6

1. Requires considerable energy in the form of ATP
2. Amino acids contribute carbon and nitrogen atoms
3. All reactions occur in cytosol
4. Highly regulated pathway
5. Erythrocytes cannot synthesize nucleotides de novo
6. Because at least 6 high energy phosphate bonds are cleaved per purine produced, salvage pathways exists to utilize preformed nucleobases and nucleosides
7. Most de novo synthesis occurs in the liver
8. A number of disease states are associated with defects in purine metabolism

Back 6

Fun facts about purine biosynthesis

RBC's can sythesize purines through salvage pathways

Front 7

1. De novo synthesis of both purine & pyrimidine nucleotides
2. The salvage of both purine & pyrimidine bases
3. The synthesis of NAD from tryptophan

Back 7

Formation of PRPP

Not the committed step, though regulated.

Front 8

Formation of N-glycosidic bond (N-9 of purine ring and C-1 of ribose)

Uses Glutamine as cofactor.

Back 8

Step 2 of pyurine biosynthesis
- Committed and regulated.
- PRPP is a feedforward activator of the enzyme

Front 9

Non-biotin dependent carboxylation.

Addition of CO2 (C-6 of purine ring)

Back 9

Step 7 of purine biosynthesis

Front 10

Cleavage of the N-C bond of aspartate.

Release of fumarate

Left with only one nitrogen

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Step 9 of purine biosynthesis

Front 11

The cell is using the other's pools in order to synthesize the other.
- Important in regulation

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Critical point of purine biosynthesis.

If you have bucketloads of GMP, want to make AMP.

Front 12

AMP + ATP -> 2 ADP

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Adenylate kinase

Front 13

GMP + ATP -> GDP + ADP

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Guanylate kinase

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__________ __________ is primarily responsible for the conversion of ADP-> ATP

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Oxidative phosphorylation

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GDP + ATP <--> GTP + ADP

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Nucleoside diphosphate kinase

Front 16

The amido transferase – ___ & ____ – the end products strongly inhibit IMP dehydrogenase and adenylosuccinate synthetase, respectively

Back 16

GMP and AMP

Front 17

Primary site: PRPP synthetase – the DPs are the major inhibitors.

Back 17

Purine regulation

Front 18

PRPP is a positive feed forward activator of the amido transferase

Back 18

Purine regulation and allosterics

Front 19

It is expensive (ATP) to make these bases, so we try to salvage them using phospho ribosyl transferases.

Base + PRPP <---> nucleoside -5’-phosphate + PPi

Adenine phosphoribosyltransferase mediates AMP formation

Hypoxanthanine-Guanine phosphoribosyltransferase mediates formation of IMP (from hypoxanthine) and GMP from guanine.

Back 19

Purine Salvage Pathways:

Free purines accumulate through the turnover of nucleic acids.

Front 20

Adenine phosphoribosyltransferase mediates ____ formation

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AMP

Salvage pathway.

Front 21

Hypoxanthanine-Guanine phosphoribosyltransferase mediates formation of ____ (from hypoxanthine) and ___ from guanine.

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IMP

GMP

Front 22

GDP, ADP are the only inhibitors.

End product inhibition

Purine biosynthesis

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Glutamine phosphoribosyl

Front 23

HGPRTase Salvage Reactions

Hypoxanthine + a-PRPP -> GMP or AMP

Guanine + a-PRPP -> GMP

Back 23

Hypoxanthine-Guanine phosphoribosyltransferase

Front 24

Uric acid is what we excrete.

Problem:
Body maintains it very close to its solubility constant

Urate not very soluble in aqueous environment.

Back 24

Uric acid is the final metabolic product in:
- Primates
- Brids
- Reptiles
- Many insects
- Dalmations

Front 25

Suicide inhibitor: A substrate for xanthanine oxidase, converted to oxypurinol – covalently modifies enzyme, resulting in a total inhibition of the enzyme activity.

Distributes endproducts of purine degradation to hypoxanthine and xanthine which are more soluble.

Still some uric acid but levels markedly reduced.

Back 25

Allopurinol

Cost: 10 to 50 cents/day

Front 26

There is a second form of xanthine oxidase that uses NAD+ as an electron acceptor.

What type of reactions is it found in?

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Detoxification reactions

Front 27

- Pyrimidine base is made first, then attached to ribose.

- UMP is precursor of all pyrimidine nucleotides with UMP giving rise to CTP and TTP.

- Salvage pathways recover preformed pyrimidine bases.

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Fun Facts about Pyrimidine Biosynthesis:

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- Different than CPS I
- Cytosolic instead of mitochondrial matrix
- Uses glutamine instead of NH3

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Carbomoyl Phosphate Synthetase II

Glutamine + CO2 + 2 ATP ---> Carbomoyl Phosphate

The first step in pyrimidine biosynthesis

Front 29

1. Aspartate trans-carbamoylase

2. Dihydroorotase
Dihydroorotate dehydrogenase

3. Orotate phosphoribosyl transferase

4. Orotate monophosphate decarboxylase

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Pyrimidine biosynthesis enzymes.

Orotic acid is the first closed form in synthesis of UMP

Front 30

To get to thiamine, must first use ______.

Back 30

UDP

Front 31

1. UDP
2. dUDP
3. dUTP
4. dUMP
5. dTMP
6. dTDP
7. dTTP

Back 31

Steps of thiamine triphosphate

Front 32

- Could end up in DNA synthesis
- Reaction must occur rapidly.

Back 32

Conversion of dUTP to dUMP

Front 33

- Methotrexate
- Aminopterin
- Trimethoprim

Back 33

Inhibit dihydrofolate reductase.

Front 34

Without dihydrofolate reductase, tumor cannot make dUTP.

Does this cure cancer?

Back 34

Constant challenge of methotrexate ramps up gene expression

Tumor designs other genes and pathways to get around competitive inhibition

Front 35

dTMP ---> dTDP (enzyme?)

dTDP ----> dTTP (enzyme?)

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TMP kinase

Nucleotide diphosphokinase

Front 36

Decides what the cell needs by:
1. Concentration of the affector
2. Affinity of the site

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Ribonucleotide reductase

ATP needs to be in active site for activity

Effector needs to be at specificity site for completion

Front 37

- Inhibited by UTP
- Activated by PRPP

As pyrimidines decrease, CPS-II is activated and synthesis increased

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CPS-II primary regulation

Front 38

As cells approach S-phase, CPS-II becomes more sensitive to PRPP activation.

Less sensitve to UTP inhibition.

Back 38

This is reversed at the end of S-phase

Front 39

- Active during cell proliferation
- Influences DNA synthesis
- Targets CPS II for phosphorylation (activates)

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MAP kinase

Front 40

Phosphorylates and inactivates CPS II

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PKA

Front 41

- Pyrimidine nucleotides are dephosphorylated

- Nucleosides are cleaved to produce ribose 1-phosphate and the free pyrimidine base.

- Cytosine is deaminated, forming uricil, which is converted to CO2, NH4+ and β-alanine.

- Thymine is converted to CO2, NH4+ and β-aminoisobutyrate.

Back 41

Degradation of the pyrimidines

Front 42

Selectively converted into acyclo-guanosine monophsophate (acyclo-GMP) by viral thymidine kinase, which is far more effective (3000x) in phosphorylation than cellular thymidine kinase.

The monphosphate form is further phosphorylated into acyclo-GTP by cellular kinase.

Acyclo-GTP is a potent inhibitor of viral DNA polymerase. It has 100x the affinity for viral than cellular polymerase

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Acyclovir

Front 43

The azido group increases the lipophilic nature of AZT, allowing it to cross the cell easily.

Thereby, crosses the blood-brain barrier.

Cellular enzymes convert AZT into 5'triphosphate form.

Termination of the formed DNA chain is the specific factor in the inhibitory effect

Back 43

AZT

Front 44

Induces apoptosis through various mechanisms.

Incorporates AraCTP into DNA and AraUMP into RNA

Inhibition of polymerase
Impairment of repair mechanisms thorugh chain termination

Besides DNA synthesis impairment, AraC engages an array of signaling events

Back 44

Cytosine arabinoside

Front 45

All carbons in the porphyrin ring are derived from glycine and succinyl CoA (all nitrogens from glycine).

Overall Rxn.
8 glycines + 8 succinyl CoA -> Porphyrin + 8CO2 + 4 NH4+

Synthesis: 80% bone marrow; 15% liver; 5% other tissues

Synthesis of heme coordinated with synthesis of globin for hemoglobin production.

Used by: Hb & Mb; cytochromes of electron transport system; cytochrome p450; catalase and others.

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Fun facts about Heme/porphyrin Biosynthesis

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All carbons in the porphyrin ring are derived from ________ and _________ ___ (all nitrogens from glycine).

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glycine and succinyl CoA

Note: succinyl CoA doesn't have any nitrogens!

Front 47

__ glycines + __ succinyl CoA -> Porphyrin + __CO2 + 4 NH4+

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8

Overall Rxn of porphyrin biosynthesis

Front 48

Synthesis: 80% bone marrow; 15% liver; 5% other tissues

Back 48

Percentages of where porphyrin synthesis occurs

Front 49

This porphyrin is used for detoxification

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Cytochrome p450

Front 50

This porphyrin is used for protein degradation.

Back 50

Catalase

Front 51

- Regulated step of heme
biosynthesis

- B6 (PLP) is cofactor

- Forms d-aminolevulonic acid from glycine and succinyl CoA

- 2 CoA and 2 CO2 leave

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ALA synthase (first step)

Front 52

Adds Fe2+ into Protoporphyrin IX.

Creates heme molecule

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Ferrochetolase

Front 53

- Represssing the synthesis of ALA synthase

- Inhibits the enzyme directly as an allosteric modifier

- Inhibits movement of ALA synthase into mitochonria

Back 53

Ways heme regulates its own synthesis.

Front 54

- In erythroid tissue, heme also regulates the synthesis of the protein globin by regulating the formation of the ribosomal initiation complex for protein synthesis.

- Iron levels will play a role in control of the translation of the ALA synthase mRNA

- The hormone erythropoietin stimulates the transcirption of the ala synthase gene.

Back 54

Other methods of regulation of heme biosynthesis

Front 55

- Absorbs UV light
- Creates free radicals
- Damage occurs at site

Back 55

Pyrrole structure.

Front 56

ALA, PBG, and their derivatives are ________ to central and peripheral nerves.

Disturbed heme synthesis in neural tissue results in depletion of essential cofactors and substrates.

Back 56

neurotoxic

For example, Schwann cells may be sensitive to damage because they synthesize and use cytochrome P-450.

Any disturbance in cytochrome production and function may lead to cell dysfunction and demyelination.

Front 57

ALA antagonizes the gamma-aminobutyric acid (GABA) receptor and may cause oxidative damage to nervous tissue.

Decreased activity of the heme-dependent protein tryptophan pyrrolase in the liver increases central and systemic tryptophan levels due to _________ tryptophan degradation.

Back 57

Decreased tryptophan degradation

Increased central 5-hydroxytryptamine levels may cause cognitive changes.

Front 58

May be caused by a combination of:
- Sustained hypertension
- Analgesic nephropathy
- Intermediates in the nephrotoxic porphyrin pathway.

Back 58

Chronic renal failure

Front 59

ALA may cause dose-dependent damage to nuclear and mitochondrial DNA.

Back 59

DNA damage

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Conjugated + Unconjugated Bilirubin =

Back 60

Total Serum Bilirubin

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Serum sample + dye =

Back 61

conjugated bilirubin (Direct)

Front 62

Serum sample + dye + alcohol =

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Total bilirubin (Total)

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Total – Direct (conjugated) =

Back 63

Indirect (unconjugated)

Front 64

A test to determine whether jaundice in a patient is due to

- Hemolysis

- Disease of the liver or bile duct.

Back 64

Serum Bilirubin Determination

"van den Bergh's test"

Front 65

High concentrations of conjugaged bilirubin indicates what disorder?

Back 65

Hemolytic problem

Front 66

High concentrations of unconjugaged bilirubin indicates what disorder?

Back 66

Liver/bile duct problem

UDP-glucaronyltransferase system deficiency

Front 67

- Excess hemolysis
- Liver damage
- Infants
- Genetic defect (Crigler-Najjar syndrome)
- Blockage of biliary tract

Back 67

Causes of Jaundace (excess bilirubin - a yellow pigment)

Front 68

1. Pyrimidine nucleotides are dephosphorylated

2. Nucleosides are cleaved to produce ribose 1-phosphate and the free pyrimidine base.

3. Cytosine is deaminated, forming uricil, which is converted to CO2, NH4+ and β-alanine.

4. Thymine is converted to CO2, NH4+ and β-aminoisobutyrate.

Back 68

Degradation of the pyrimidines:

Front 69

Inhibits the amidotransferase

Back 69

6-mercaptopurine 5’monophosphate

Front 70

Irreversibly (suicide) inhibits thymidylate synthase

Back 70

5FdUMP

Important agent in treatment of breast and colon cancer.

Front 71

1. Orotate phosphoribosyl transferase
2. OMP decarboxylase

What multifunctional enzyme is it?

Back 71

UMP synthase

Affected by Hereditary orotic aciduria

Front 72

1. Pyrimidine nucleoside phosphrylase (non-specific)converts the pyrimidine bases to their nucleosides.

2. Specific nucleoside kinases then react with the nucleosides, forming nucleotides.

Back 72

Salvage of pyrimidine bases

Pyrimidine phosphorylase has a preferance for uracil, uses cytosine sometimes, and rarely uses thymine.

Front 73

The activity of _____ rises dramatically as the cell enters S-phase.

Enzyme is allosterically inhibited by dTTP.

Back 73

Thymidine kinase (TK), a ribonucleotide kinase.

Radiolabeled thymidine is widely used for isotopic labeling of DNA

Can estimate rates of intracellular DNA synthesis.