What is phosphoribosylpryophophate (PRPP)
It must be made in order to run both the purine and pyrimidine de novo syntheses
How is PRPP created?
In the reaction of 5-phophoribose with ATP to create it
How is the PRPP synthesis regulated?
Its very highly regulated Its activated by the presence of high levels of inorganic phosphate Its inhibited by nucleotides such as ADP, IMP, etc
Which de novo pathway starts with PRPP?
Purine de novo synthesis starts with it and builds the nitrogenous bases on the sugar
How does the pyrimidine de novo synthesis use PRPP?
It builds its nitrogenous bases and then attaches them to the PRPP
Is PRPP considered a step in either de novo pathway?
NO!! it is just used as a ribos sugar and during the two de novo pathways bases are added onto it
What is the first nucleotide synthesized in the purine de novo synthesis?
Inosine monophophate (IMP)
What is the first nucleotide synthesized by the pyrimidine de novo synthesis?
Uridine monophosphate (UMP)
What is the first step in the purine de novo pathway?
PRPP reacts with glutamine to generate phosphoribosylamine (PRA) ****This is the committed step; meaning you can stop it until IMP is formed Its also the rate limiting step; due to the feedback inhibition caused by AMP and GMP
What is the highest regulated step in the purine de novo pathway?
The first test of PRPP reacting with glutamine to generate phosphoribosylamine (PRA) Due to the feedback inhibition of AMP and GTP
In purine de novo pathway when is IMP converted into AMP?
When you can use GTP as the energy source. MORE GTP means you get more AMP from pathway
In the purine de novo pathway when is IMP converted to GMP?
When you use ATP as the energy source. More ATP means more GMP production
What are the two places for regulation in the purine de novo pathway?
1. PRA synthesis (inhibiting glutamine-PRPP amidotransferase) 2. Conversion of IMP to either AMP or GMP
How does PRA synthesis during purine synthesis get inhibited?
The more production of GMP and AMP you get the more feedback inhibition you get. GMP and AMP block the reaction of PRPP with glutamine so you do not create PRA
How does the conversion of IMP to either AMP or GMP effect the purine de novo pathway in terms of regualtion.
The more you create of one the more it inhibits its production AMP will stop production of more AMP GMP will stop production of more GMP In reverse tho the more GMP you make the more it will drive the rxn to make more AMP and vis versa
Since the PRPP has one phosphate group how do you add more phosphates to the nucleotides?
Use very specific enzymes Nuleoside monophophate kinases (NMPK) adds 2nd phosphate Nucleoside diphosphate kinases (NDPK) adds third phosphate
What is an NMPK?
Its a nucleoside monphosphate kinases and adds a second phosphate to the nueculsides for ex: AMPK - adaine monophosphate kinases adds it to the alanine group and the others follow suit CMPK GMPK TMPK UMPK
What is the first step in the pyrimidine de novo pathway?
The formation of carbamoyl phosphate and it the most highly regulated step
What is the most highly regulated step of the pyrimidine de novo pathway?
The first step of the formation of carbamoyl phosphate Its activated by ATP and PRPP Its inhibited by CTP
What is the second step of the pyrimidine de novo pathway?
The formation of N-carbamoyl aspartate This is also the committed step
What is the committed step of the pyrimidine de novo pathway?
The formation of N-carbamoyl aspartate Once this step occurs you will get UMP formation Also the rate-limiting step (most susceptible to feedback inhibition)
How is UMP converted to CTP?
1. UMP is first convered to UTP thro nuceotide kinases UMPK and UDPK 2. UTP is then converted to CTP in a one step rxn 3. Glutamine donates the amine and with ATP it creates the CTP
Where does the primary regulation of the pyrimidine synthesis take place and how is it inhibited?
At carbamoyl phosephate synthetase which carries the synthesis of carbamoyl phosphate This is inhibited by CTP; the end product of the pathway CTP also inhibits the production of more CTP from UTP
What are salvage pathways?
When you get base excisions or when purines spontanously break free you get free purine and pyrimidine bases that can be used
Can the salvage pathway inhibit de novo pathways?
Yes, it does inhibit it It lowers the amount of PRPPs and raises the levels of NTPs (nucleotides)
How does the salvage pathway inhibit the purine pathway?
It adds bases to PRPP which decreases the amount of PRPP to be used in the pathway Hypoxanthine-guanine phophoribosyltransferase (HGPRT) this adds an insoine base to a PRPP Adenine Phosphoribosyltransferase (APRT) adds an adenine to a PRPP
What does Hypoxanthine-guanine phophoribosyltransferase (HGPRT) do?
This adds an inosine base to a PRPP which will decrease the amount of PRPPs available and inhibits de novo pathways **this is a salvage pathway
What does Adenine Phosphoribosyltransferase (APRT)
adds an adenine to a PRPP which will decrease the amount of PRPPs available and inhibits de novo pathways **this is a salvage pathway
How does the salvage pathway affect the pyrimidine pathway?
Pyrimidine phosphoribosyl transferase can salvage orotate, uracil and thymine
What can be done in order to salvage cytosine?
Nothing can do it directly but it can be deaminated to uridine which would allow it to enter the salvage pathway and be broken down
What can happen during purine degradation that can lead to uric acid?
Most ingested nucleotides are degraded to ribose, which is converted to energy and the nitrogenous base Purines that are not salvaged this way are degraded directly and create uric acid by the enzyme xanthine oxidase
What happens when uric acid crystallizes?
It turns into crystals in the joints and this is the cause of gout. The crystals poke and jab you in the joints to give a lot of pain.
How does the body create deoxyribonucleotides?
The enzyme ribonucleotide reductase (RNR) converts NDPs (ribonucleoside 5’-diphosphates) into dNDPS (deoxyribonucleoside diphophate)
What is required of RNR?
Is non-specific and requires thoredoxin, a small protein that contains two cysteins residues which form disulfide bonds
How is Thymidine created?
It used Thymidylatye synthase and the use of dUMP
How does thymidylate synthase work?
Its an enzyme that catalyzes the transfer of one carbon from 5,10-methylene-tetrahydrofolate to dUMP This addition of the metyhl group to dUMP creates dTMP
What is dihydrofloate reductase (DHFR)
Its what is required to create make 5, 10-methylene-tetrahydrofolate which is a compound used in the synthesis of dTMP for dUMP and synthesis of DNA
What is required for the synthesis of DNA specifically regarding the synthesis of dTMP from dUMP
You need both thymidylate synthase (which takes the methyl from the 5,10 thingy and adds to the dUMP) You also need DHFR because it will create the 5,10 methylene **NEED both of these for DNA synthesis
What are Antimetabolites?
They are compounds that resemble and interfere with something required for metabolism
What are some examples of antimetabolites?
5-fluorouracil and capecitabine which resemble dUMP and inhibit thymidylate synthase 6-mercaptopurine, an analog of hypoxanthine (leads to xanthine) Allopurinol - a substrate and then inhibitor xanthine oxidase
What are antifolates?
These resemble a portion of tetrahydrofolate
What are some examples of antifolates?
Aminopterin, methotrexate, and trimethoprim which inhibit dihydrofloate reductase Sulfa antibiotics which block the synthesis of folic acid
How does allopurinol and febuxostat treat gout?
Remember Xanthine oxidase carries out the last two steps in the formation of uric acid 1. Allopurinol and febuxostat inhibit xanthine oxidase 2. if you inhibit xanthine oxidase it will decrease the amount of uric acid in the system
What specifically does allopurinol do to xanthine oxidase?
It makes oxypurinol (adds an oxygen) which inhibits the use of the enzyme
How does 5-Fluorouracil and capecitabine (prodrug) inhibit dTMP synthesis?
They act as suicide inhibitors and covalently bind to and irreversibly inhibits thymidylatye synthase **adds a F which can not be taken off
How does Aminopterin Methotrexate and Trimethoprim stop dTMP synthesis?
It inhibits dihydrofolate reducatse **remeber this creates the 5, 10 methylene thing that is used in order to convert dUMP to dTMP
How does bactrim (trimethoprim + sulfamethoxazole) stop dTMP synthesis?
It inhibits both folic acid synthesis and dihydrofolate reductase
What two enzymes are used in the salvaging nucleosides?
Deoxycytidine kinase Thymidine kinase **these are very specific and add the first phosphate to recover the nucleoside **they only work on cytidosine and thyosine
How does Acyclovir and Valacyclovir work as antivirals?
They resemble guanosine which means they act like nucleoside reverse transcriptase inhibitors (NRTIs) *it gets phosphorylated and activated then gets incorporated into viral DNA which stops it ably to replicate and it dies **Important to know this only slows down infection and eases symptoms, you body still needs to deal with it