Roth

Question 1

What are the purines?

Answer 1

AG

• Pure Gold (Ag = gold)
• 2 ring structure

Question 2

What are the pyrimidines?

Answer 2

Cytosine, Uracil, Thymine

- 1 ring structure

Question 3

What is the difference between a nucleoside and nucleotide

Answer 3

Nucleoside is just the base and the ribose

Nucleotide is the base, ribose, and the PHOSPHATE

Question 4

What is the first step in de novo purine synthesis?

Answer 4

First step is to make PRPP.
Ribose-5phosphate –> PRPPP

• the enzyme that does this is called “PRPP Synthetase”
• One molecule of ATP is consumed
  Regulation:
  Upregulated - by inorganic phosphate because if you remember, inorganic phosphate is released any time a nucleotide is destroyed/recycled,, signaling a need for more to be made
  Down regulation - ADP, ATP, GDP, GTP.

Question 5

WHat is the second step of de novo purine synthesis after we have PRPP?

Answer 5

PRPP –> PRA

• this is the committed step in purine synthesis
• Completed by the enzyme “PRPP-amido-transferase”
• Glutamine donates the amide group for this to be able to occur.

Regulation:
Up regulation - PRPP
Down regulation - AMP, GMP, IMP

Question 6

Azaserine and DON

Answer 6

Structurally similar to glutamine. They irreversibly bind to PRPP-amide-transferase. These antibiotics are potent inhibitors or purine nucleotide metabolism.

Question 7

What is the 3rd step in de novo purine synthesis? Now that We have PRA, what happens?

Answer 7

There is a series of 9 steps that converts PRA into IMP.

• 4 molecules of ATP are used up in this process
• contributors towards making IMP are: CO2, aspartate, glycine, ,glutamine, THF

Question 8

How do humans make THF?

Answer 8

Dietary folic acid –> dihydrofolate –> THF

• DHF reductase is responsible for the conversion of dihydrofolate into THF.
• This enzyme is heavily acted upon by many drugs as a chemotherapeutic agent.

Question 9

How does methotrexate / aminopterin work?

Answer 9

They bind to DHF reductase, which stifles nucleotide synthesis and kills tumors. It is very toxic though

Question 10

How does trimethoprim work?

Answer 10

Another drug that inhibits DHF reductase. This one though binds much stronger to bacterial DHF reductase than human DHF reductase. Therefore, it is an effective antibiotic.

Question 11

How do sulfonamides work?

Answer 11

Basically, bacteria make their own folate from a molecule called PABA. Sulfonamides are structurally similar to PABA and therefore competitively inhibits folic acid synthesis.

Question 12

Step 4 in de novo purine synthesis

Answer 12

IMP can be converted into AMP or GMP downstream. This slide discusses AMP synthesis

IMP is converted into the purine intermediate adenylosuccinate.

• GTP and aspartate are needed for this step
• The enzyme that does this is inhibited by AMP

Then, adenylosuccinate is converted to AMP, releasing fumarate in the process.

Question 13

Step 4 in purine de novo synthesis

Option2

Answer 13

The other option for IMP is to be converted into GMP. First, IMP is converted into XMP by IMP dehydrogenase.

• NAD+ is required for this reaction.
• IMP dehydrogenase is inhibited by GMP

Secondly, XMP is converted to GMP.

Question 14

Ribavarin

Answer 14

Antiviral medication used to Hep C. it inhibits IMP dehydrogenase, thereby depleting intracellular pools off guanine nucleotides.

Question 15

Regulation of AMP and GMP

Answer 15

They both feedback on their own creation via negative feedback.
They are also regulated so as to coordinate the relative amounts of AMP and GMP to one another in a process called reciprocity.
- This basically means that if you remember, you need GTP to stimulate the creation of AMP and you need ATP to stimulate the creation of GMP. Therefore, if levels of one are low, the other will ensure that it gets more made.

Question 16

Step 5 of de novo purine synthesis? WHat happens once we have our AMP/GMP?

Answer 16

We’ll they eventually have to be made into ATP/GTP. So, first, an enzyme called Monophosphate kinase that produces ADP and GDP. ADP and GDP are further phosphorylated by nucleoside diphosphate kinase to create ATP and GTP.

Question 17

Step 1 in purine salvage pathway

Answer 17

The salvage pathway takes advantage of the fact that free floating bases including, adenine, guanine, and hypoxanthine are free floating in the cell. The salvage pathway simply attaches a preformed purine base to the PRPP backbone. There are two enzymes that do this:
APRT converts: Adenine + PRPP –> AMP
HGPRT converts: Guanine + PRPP –> GMP
HGPRT also converts: Hypoxanthine + PRPP –> IMP

Question 18

Lesch Nyhan Syndrome

Answer 18

Caused by a deficiency in the enzyme HGPRT

• X linked recessive
• results I n neurological defects such as spasticity, mental retardation, aggression, self-mutilation.
• Because you have less HGPRT, you have less GMP and thus less GTP. The brain relies very heavily on this pathway, which is why we see the neurological deficits. It is even involved in the developmental process of dopaminergic neurons, which is why these patients have less dopaminergic nerves.
• Because the PRPP isn’t being used to make GMP/IMP, there is also extra PRPP laying around. This increases purine de novo synthesis because it is an activator off this pathway (PRPP-amido-transferase).
• These patients also have uric acid buildup resulting in gout. This makes sense because you have extra guanine and hypoxanthine. These bases will then become uric acid.

Question 19

What is the first step in the degradation of purines to uric acid?

Answer 19

A 5’ nucleotidase removes the phosphate group from AMP, GMP, IMP, resulting in the formation of adenosine, guanosine, and inosine. What is left s just the nitrogenous base attached to ribose.

Question 20

What is the Second step in purine degradation?

Answer 20

Adenosine is converted into Inosine using an enzyme called “Adenosine Deamine (ADA)”.
Deficiency of ADA causes a disease called SCID

Question 21

SCID - severe combined immunodeficiency

Answer 21

Deficiency in ADA, causing severe lack of B and T lymphocytes that predispose affected patients to bacterial, viral, and opportunistic infections early in life that are often fatal.
- usually treated with a bone marrow transplant.

Question 22

What is the third step in purine degradation? Once we have our inosine

Answer 22

So at this Point, we have Inosine and guanosine. What we do here is remove the ribose sugar from Inosine and guanosine in order to leave their respective nitrogenous bases, hypoxanthine and guanine.

• This is done by an enzyme called purine nucleoside phosphorylase (PNP).
• PNP mutation would cause another subtype of SCID called “PNP Deficiency”

Question 23

PNP Deficiency

Answer 23

A subtype of SCID, which occurs due to a mutation in the PNP enzyme.
- Characterized by a T-cell deficiency. It predisposes patients to bacterial, viral, and opportunistic infections. It usually presents in childhood.

Question 24

What is the 4th step in purine degradation? Once we have guanine and hypoxanthine what happens?

Answer 24

They are both converted into xanthine by various enzyme.

• The enzyme that converted hypoxanthine is called “xanthine oxidase”
• this is the conversion point from the degradation of AMP, GMP, and IMP.

Question 25

WHat is the 5th step in purine degradation? Once we have our xanthine what happens?

Answer 25

Xanthine oxidase, the same enzyme that converted hypoxanthine to xanthine, now converts xanthine into uric acid, which is then excreted into the urine.

Question 26

Gout

Answer 26

When uric acid levels get too high, beyond their solubility limit, insoluble crystals of sodium rate deposit throughout the body's tissues.

Question 27

Primary hyperuricemia

Answer 27

Causes by an inborn error in uric acid metabolism resulting in overproduction of uric acid.

Question 28

Secondary Hyperuricemia

Answer 28

Caused from an accumulation of uric acid due to another disease such as a malignancy, chronic renal insufficiency, G6P deficiency, and some meds.

Question 29

How is gout treated and why does it work?

Answer 29

Treated with allopurinol - Mechanism has a 3-pronged attack: 1) It is an analog of hypoxanthine and inhibits xanthine oxidase. 2) Xanthine oxidase then converts allopurinol into alloxanthine, a potent irreversible inhibitor of xanthine oxidase. 3) It also leads to a buildup of hypoxanthine, which can be salvaged to produce IMP, which serves as an inhibitor of PRPP--amido-transferase. - allopurinol can also act as a substrate for HGPRT, which converts it into a ribonucleotide. This then lowers the levels of PRPP, which lowers the amount of PRPP available for de novo synthesis. This also causes decreased feed forward feedback so less de novo synthesis will occur.

Question 30

Purine Nucleotide cycle

Answer 30

If you remember, earlier in the synthesis of de novo purines, we saw that IMP gets converted to AMP. This conversion releases fumarate, which is a key TCA cycle intermediate. IMP can also then be converted back to AMP in the degradation cycle. This is called a "anaplerotic reaction" because it serves to replenish the intermediates of another metabolic process.

Question 31

What is the first step in de novo pyrimidine synthesis?

Answer 31

2 ATP + CO2 + Glutamine --> Carbamoyl Phosphate - The enzyme responsible for this is called "Carbamoyl Phosphate Synthetase II" Regulation: - up regulation - ATP and PRPP - down regulation - UTP, the end product of the pathway

Question 32

What is the second step in de novo pyrimidine synthesis? Once we have our Carbamoyl phosphate what happens?

Answer 32

There is a ring closure Into something called "Orotate". The Carbamoyl phosphate only contributes two atoms to this ring though, the other 4 atoms come from aspartate. The enzymes that do this as well as Carbamoyl phosphate are all different domains of one large polypeptide called CAD.

Question 33

What is step 3a in pyrimidine de novo synthesis? Once we have Orotate what happens?

Answer 33

Orotate + PRPP --> OMP - This is done via the enzyme Orotate phosphoribosyl transferase - OMP is the precursor to the parent precursor, UMP.

Question 34

What is Step 3b in the de Novo pyrimidine pathway?

Answer 34

OMP --> UMP - this is done via the enzyme orotidylate decarboxylase - Ototidylate decarboxylase and Orotate phosphoribosyl transferase are two domains of one larger polypeptide.

Question 35

Orotidylate aciduria

Answer 35

Decreased activity of orotidylate decarboxylase or Orotate phosphoribosyl transferase will cause this. - will cause megaloblastic anemia and Orotate in the urine. - this is not corrected with Vitamin B12 and folate but rather you end CMP, UMP, or uridine in order to bypass the block and treat the disease.

Question 36

What is step 4 of de novo pyrimidine synthesis? Once we have UMP what happens?

Answer 36

UMP is phosphorylated to UDP by Monophosphate kinase. | UDP is then converted to UTP via nucleoside diphosphate kinase.

Question 37

WHat is step 5 of pyrimidine synthesis? Once we have UTP what happens?

Answer 37

UTP --> CTP This occurs via an enzyme called CTP Synthetase. - requires ATP and glutamine. - CTP then negatively inhibits this rxn.

Question 38

How is Thymine made?

Answer 38

2 ways: 1) from a precursor molecule called dUMP - dUMP is converted into thymidylate via the enzyme thymidylate synthase in the presence of N5-N10-methylene THF. 2) from the deamination of dCMP

Question 39

How are deoxyribonucleotides made?

Answer 39

Ribonucleotide Reductase Ribonucleotide diphosphate --> deoxyribonucleotide diphosphate - done via the enzyme ribonucleotide reductase. - Uses NADPH so that the active site can be reduced. Regulation: Up - ATP Down - dATP

Question 40

5-fluorouracil

Answer 40

Antitumor agent that gets converted into 5-FdUMP, which inhibits thymidylate synthase. This blocks dUMP conversion to TMP.

Question 41

Salvage of pyramidines

Answer 41

Requires 2 steps: 1) A pyrimidine base and a ribose-11-phosphate are converted to pyrimidine Ribonucleoside and inorganic phosphate. 2) Pyrimidine nucleoside is phosphorylated to become pyrimidine nucleoside Monophosphate by an enzyme, nucleoside kinase. - one of these kinases is called thymidine kinase, which salvages thymidine to TMP.

Question 42

Acyclovir

Answer 42

Because Herpes simplex virus encodes its own thymidine kinase, it is able to augment its own replication. - acyclovir, a guanosine analog, interferes with viral replication by being incorporated into viral DNA, resulting in premature DNA chain termination.

Question 43

Purines de novo synthesis | Pyramidine de novo synthesis

Answer 43

Purine - synthesizes parental purine from scratch ON a ribophsphate backbone Pyrimidine - synthesizes the parental backbone from scratch and then ATTACHES it to a ribophsphate backbone

Question 44

Mycophenolic acid

Answer 44

Blocks T cells and B cells of nucleotides and is therefore used as an immunosuppressant for grafts.

Question 45

What are the names of the kinases that convert AMP/ GMP to ADP/GDP and eventually to ATP/GTP?

Answer 45

The first kinase is base specific, so it is called adenylate kinase and guanylate kinase. The second kinase is not base specific and is called nucleoside diphosphate kinase

Question 46

What is the name of the kinase that converts UMP-->UDP and what converts UDP-->UTP?

Answer 46

UMP --> UDP = nucleoside mono phosphate kinase. This is base specific for UMP UDP-->UTP = nucleoside diphosphate kinase. This is not base specific and will convert any nucleoside diphosphate into any nucleoside triphosphate

Question 47

How does ribonucleotide reductase know which deoxyribonucleotide to make?

Answer 47

Depending on what is bound to the "specificity site" will affect which ribonucleotide fits in the catalytic site to get converted into deoxyribonucleotide.

Question 48

What is the proposed mechanism for how ADA deficiency causes SCID?

Answer 48

With the inability to convert adenosine to inosine, you have a buildup of adenosine. This will then be made into ribonucleotides and deoxyribonucleotides. The buildup of dATP will cause inhibition of ribonucleotide reductase and the inability to make deoxyribonucleotides and thus no cell division (specifically for lymphocytes).

Question 49

We said that one of the ways TMP can be made is via dUMP. How is dUMP made though?

Answer 49

Either from dUTP or dCMP.