Purine Metabolism

Last time we talked about the structure of different Purines and Pyrimidines, and we started talking about the synthesis of these nucleotides (purien nucleotides) and you saw the pathways for the synthesis of purines; there are two pathways:

1- De Novo pathway:
De novo as you know stars from the scratch, it will start from the ribose 5-phosphate which doesnt have purine nitrogen bases, and goes to phosphoribosel pyrophosphate (PRPP) which will react with glutamine glycine, one carbon pool (N10-formyl THD), another glutamine,HCO3 -, aspartate and another one carbon pool (N10fromyl THF) till we reach to the IMP ( Inosine MonoPhosphate ) which is considered the parent compound for the synthesis of AMP and GMP. Then AMP and GMP will be phospholyrated to give ATP and GTP. This is called the De novo (Latin: a new) pathway for purine biosynthesis; that will start from non purine precursor such as ribose and amino acids to give the purine nucleotides.

2- Salvage pathway:
The salvage pathway is abundant in some specific tissues and it uses purine themselves as precursors, that will combine with PhosphorRibosyl PyroPhosphate (PRPP) to form the corresponding purine nucleotides, this is an enzyme catalyzed reaction by Hypoxanthine Guanine PhosphoRibosyl Transferase enzyme (HGPRT) that will form the GTP (if the purine precursor is Guanine

or Hypoxanthine) or by another enzyme (APRT) that will form the ATP (if the purine precursor is adenine).

Catabolism of Purines:
Now this is AMP and this is the GMP that will be converted to ATP and GTP, upon some conversion or changes on these purine nucleotides, as you see IMP gives AMP and also AMP gives IMP. So if you deaminate AMP you will get IMP and IMP will be converted to Hypoxanthine , some more reduction and Hypoxanthine will be oxidized by an enzyme called Xanthine Oxidase , GMP also could be converted to xanthine , now the xanthine that comes from AMP and GMP will be oxidized by Xanthine Oxidase to give Uric Acid, the massage is: if you have excess of purine nucleotides (AMP or GMP) all the excess will be converted to Uric Acid and Uric Acid if it exceeds it's physiological concentration it will cause diseases, it will cause what's called GOUT .

Now if Uric acid exceeds it's physiological concentration , because of excess of AMP and GMP synthesis it will be precipitated (this Uric acid) in the form of Urate in the joints (especially in the extremities like the fingers), that will cause a lot of pain , and this is called hyperuricemia which is the GOUT. Last time I talked about regulation and the control, nothing in the cell takes place haphazardly, every thing is under control, metabolic pathways are highly regulated and here we have a regulation; when we have excess of AMP or excess of GMP, we have what's called the feedback inhibition, AMP will inhibit the enzyme that converts IMP to AMP, GMP will inhibit the enzyme that converts IMP to GMP. also these AMP , GMP , IMP will inhibit the phosphoribosyl pyrophosphate synthetase , so there is a good regulatory mechanism, but if we have some mutations, gene mutations concerning the phosphoribosyl pyrophosphate synthetase or the enzyme that is responsible for the salvage pathway , which is hypoxanthine guanine phosphoribosyl transferase , if we have mutations on those genes that are responsible for these two enzyme then we will have excess purine nucleotides and thus we have hyperuricimea . This is the gout which I define it as hyperuricimea, high concentration of the uric acid above the physiological conditions in the blood and that could result from many causes.

Causes that lead to hyperuricimea:

The first one is over production in the de novo pathway (started from scratch, non purine precursor) so over production will lead to hyperuricimia.

The second is kidney damage resulting in a failure excretion of uric acid, if there is a problem in the kidney excretion will be ineffective and thus uric acid will be highly precipitated as urate and crystallized in the joints.

The third is mutations in the HGPRT (Hypoxanthine Guanine Phosphoribosyl Transferase) which is the principle enzyme for the salvage pathway, so mutation in this enzyme and also mutation in PRPP synthetase enzyme (the fourth reason) will lead to Hyperuricemia. Over production in the de novo pathway of purine nucleotide will lead to Hyperuricimea because we have excess AMP, IMP and GMP, then AMP will be converted to Hypoxanthine and GMP to Xanthine, then both will be converted to Uric acid by Xanthine Oxidase enzyme. Why mutation in the HGPRT enzyme will lead to Hyperuricemia? This enzyme combines PRPP with guanine to get GMP, and another enzyme replaces the adenine instead of guanine so it will make AMP in the salvage pathway. If this enzyme becomes mutant then we will have accumulation in it's substrate, which PRPP mainly, PRPP is also used in the de novo pathway, now the high amounts of PRPP will be used in the de novo pathway to produce excess AMP and GMP that will be converted to Uric acid. This gout or Hyperuricimea that resulted from these factors is called PRIMARY GOUT.

There is another type called SECONDARY GOUT , and that results because of deficiency of an enzyme called Glucose 6- phosphatase , its function is to convert glucose 6-phosphate to glucose , so if we have deficiency in this enzyme then we will have excess or accumulation of glucose 6-phosphate , then glucose 6-phosphate will be directed into hexos monophosphate shunt or pentose phosphate pathway, that will be converted to ribose 5-phosaphate , ribose 5phosphate is the precursor for phosphoribosyl pyrophosphate (PRPP) , phosphoribosyl pyrophosphate is the proper substrate for de novo and salvage , so excess of uric acid will be produced as result of deficiency of glucose 6-phosphatase.

If we have partial deficiency of HGPRT, the patient will have Hyperuricemia that leads to Gout, but if the patient has complete deficiency of HGPRT due to mutations that are responsible to produce this enzyme, hyperuricimea will result, but in this case because of high excess of uric acid upon the complete deficiency of the HGPRT excess uric acid that will result in a syndrome called Lesch - Nyhan Syndrome, this is an " X " - linked syndrome , Xlinked disease and it's a neurological disease and patients that have complete deficiency of this enzyme , will have mental retardation and

self humiliation , because the salvage pathway was found to be dominant in brain tissue in the nervous system (the Central Nervous System). Though, excess of uric acid in the brain will lead to damage of the brain that will cause this type of mental retardation. So partial deficiency of HGPRT will lead to GOUT, whereas complete deficiency will lead to Lesch Nyhan Syndrome, which is an X-linked neurological disease.

How do we treat Gout? How to stop the formation of uric acid? By inhibiting the xanthine oxidase enzyme. This is a diagram of a drug called Allopurinol and this drug is used in the treatment of Gout.

It's action is to inhibit Xanthine Oxidase, that converts Hypoxanthine to Xanthine then to Uric acid, Allopurinol itself is not active so it must be activated in order to be effective in inhibiting Xanthin Oxidase.

How is Allopurinol activated? By Xanthine Oxidase itself, it will be converted to Alloxanthine, Alloxanthine is the principle drug or compound that inhibit Xanthine Oxidase. So Xanthine Oxidase causes inhibition of itself, by activating Allopurinol and converted to Alloxanthine then Alloxanthine start to inhibit the enzyme that activates it, because of this, it's called a suicide inhibitor. This is the end of purine metabolism. :D

Pyrimidine Metabolism
We will start from glutamine, glutamine is an acid, and we need glutamine as a nitrogen source for pyrimidine synthesis plus ATP plus CO2 by an enzyme called Carbamoyl phosphate synthetase, Carbamoyl phosphate will be produced as follows:

This reaction is catalyzed by the enzyme Carbamoyl Phosphate Synthetase II (CPS-II)

Carbamoyl phosphate plus Aspartate gives N-Carbamoylaspartate:

This enzyme is very important, you have know it and you have to remember it, because it's the key enzyme for pyrimidne biosynthesis, if you remember from your metabolism course, in urea cycle, one of the important enzyme to start urea synthesis is carbamoyl phosphate synthetase also, and here we have carbamoyl phosphate synthetase but in order to differentiate them we have carbamoyl phosphate synthetase (I) for pyrimidine . And carbamoyl synthetase (I ) for urea cycle .

The difference between carbamoyl synthetase () and () (The doctor said that synthase and synthetase are the same)

You are not suppose to memorize these reactions, but I want you to remember for pyrimidine biosynthesis we started with carbamoyl phosphate and Aspartate and with few other steps to form what is called orotate . Orotate is the parent compound for pyrimidine nucleotides, while IMP is the parent compound for purine nucleotides. This orotate combines with PRPP, while in purine biosynthesis we started with PRPP amide then different amino acids like glycine, glutamine and Aspartate, one carbon pool came after PRPP while here we form the parent compound of pyrimidine nitrogen base as orotate, then PRPP combines to it to form the orotidylate (pyrimidine nucleotide) because Orotate is a nitrogenous base. Pyrimidine nitrogen base becomes a nucleotide, then some modification and decarboxylation occurs, converting orotidylate to UMP (uridylate).

Orotate comes from carbamoyl phosphate with aspartate after some biochemical reactions it becomes orotic acid (orotate ) then PRPP converts this pyrimidine nitrogen base to pyrimidine nucleotide (uridylate) , uridylate by further modification or decarboxylation enzymatic reactions is converted to UMP, so this is the case in which pyrimidine nucleotides are formed, now UMP by further phospholyration it becomes UTP, then UTP will be converted to CTP. Uridylate (UMP) and Cytidilate (CMP) are formed in this pathway and in other reduction reaction you are going to see how thymidilate is synthesized. Orotic aciduria: a genetic disease that causes mental retardation; the uric acid will be very high in the uria, the cause behind aciduria is the accumulation of the orotate in the uria.

The two enzymes that are responsible to convert orotic acid to orotodilate and uritidylate and cytidylate are blocked or defected because of mutations. Treatment: Replacement of uritidylate (UMP) and cytidylate (CMP). How is UTP converted to CTP?

UMP to UTP (by phosphorylation) then UTP to CTP (by amidation) and this is enzymatic here , it requires amidation of UTP to convert it to CTP and the amino group will be taken from glutamine given to uridine and you will convert uridine to cytidine ,this is how CTP is formed.

Now we know how AMP, GMP, UTP and CTP are synthesized but what do we need to synthesis DNA? We should have the deoxy forms of these nucleotides.

How do we convert them to the deoxy forms? By ribonucleotide diphosphate reductase, it's very important to convert the ribonucleotide diphosphate to deoxy ribonucleotide diphosphate and it's a very dangerous enzyme (that is under regulation) and if we inhibit this enzyme there will be no life, no DNA synthesis, no genes and no chromosomes. This is the big enzyme; this is the Ribonucleotide reductase enzyme that converts ribonucleotide diphosphate to deoxyribonucleotide diphosphate. The substrate is ribonucleoside diphosphate + thioredoxin. Thioredoxin (reduced form) is a coenzyme for this enzyme and this is

the first time that we have a coenzyme which is a protein, usually coenzymes are organic compounds but not proteins, this is the first case in which co-enzyme is a protein for this enzyme so ribonucleoside diphosphate +thioredoxin will convert the ribonucleoside to 2'-deoxyribonucleoside diphosphate and this is what we need for genes or DNA synthesis.

Indeed we don't want, deoxynuclesoside diphosphat we want triphosphate nucleoside for the DNA synthesis, so it must be converted later to triphosphate. Thioredoxin ( oxidized form) must be regenerated in the presence of FADH2 which will be oxidized to FAD, in turn FAD will be reduced back to FADH2 and NADPH will be oxidized to NADP+ to regenerate FADH2 in order to complete this reaction. So as you see the enzyme requires 3 Coenzymes; Thioredoxin which is a protein, FAD and NADPH. TMP is synthesized from deoxyuridinate monophosphate (dUMP), and we see dUDP after phosphlyration of dUMP. dUMP by the enzyme thymidilate synthase will be converted dTMP and this is what we want for the DNA indeed .

We want dTTP but this dTMP is easily converted by two successive phospholyrations to dTTP. Now this enzyme thymidilate synthase (dont forget this enzyme, it's a very important enzyme) without it we can't get dTMP and without deoxythymidine nucleotide we can't replicate our DNA at all. So life will stop without this enzyme , this enzyme requires N5, N10 Methylene tetrahydrofolate (one carbon pool) so you are going to see how this one carbon pool will be provided and reduction by two hydrogens given to dUMP in order to convert to thymidilate. Remember that thymidilate in the structure it has methyl group, and you have to remember that thymdiliate synthase required the Coenzyme (N5 , N10 Methylene tetrahydrofolate ) in order to get dTMP. Regulation or inhibition of this enzyme could control or stop growth of cancer cells and here a lot of drugs nowadays are use to inhibit thymidilate synthase, and this is a strategy of cancer treatment by finding drugs that will inhibit thymidilate synthase and stop this reaction in cancer cells.

Regulation of ribonucleotide diphosphate reductase:

Why we don't have TDP?! Because it has already been converted by thymidilate synthase. Thymidilate is not a substrate here, because we have specific pathway for synthesis of thymidilate. There are positive and negative effectors for this enzyme:

In this enzyme with all substrates we have dATP as a negative effector, so dATP is a very dangerous compound, if it's in high concentration it will be highly toxic to the cell, we have some gene infection, that will lead to high concentration above physiological

concentration of dATP and that will lead to a very serious genetic diseases.

Tetrahydrofolate is important, it has one carbon pool for many properties: - De novo biosynthesis of purine. Thymidilate synthase Coenzyme. So deficiency of folate will be very dangerous, so you have to keep up with folate level in your body, because we couldn't synthesis it in our body. Dihydrofolate will be converted to tetrahydrofolate by to successive reductions and that require NADPH and enzyme called Dihydrofolate reductase . Dihydrofolate reductase is also a very important enzyme, because it will provide Tetrahydrofolate, Tetrahydrofolate is important for Thymidilate synthase.

Deficiency of this enzyme (Dihydrofolate reductase) will lead to genetic diseases, if the gene itself is defected. There are some compound that reassemble in structure the folate specially this active center of this compound (picture above), if those chemicals reassemble this center of the compound then they could act as competitive inhibitors, so some drugs that have similar structure of this in folate like a drug called methotrexate (MTX) and it's for cancer treatment, it will cause inhibition of Dihydrofolate

reductase. And if dihydrofolate is inhibited, we don't have tetrahydrofolate, then thymidilate synthase will stop, and thus no DNA synthesis so death to cancer cells.

This is the pathway of how MTX act as anticancer drug :

And this is how dUMP converted to dTMP it requires tetrahydrofolate in order to be converted:

Once dUMP is converted to dTMP tetrahydrofolate will be converted to dihydrofolate , tetrahydrofolate must be regenerated from dihydrofolate , dihydrofolate is converted to tetrahydrofolate by dihydrofolate reductase , MTX will inhibit this, that means there is no conversion of dUMP to dTMP. Adenosine deaminase: deaminating Adenine or deoxy adenosine converts it to Inosine or deoxyInosine. If this enzyme is deficient then you will have accumulation of adenosine or deoxyadenosine and those adenosine or deoxyadenosine will be used if accumulated to synthesize dATP.

What is the effect of high concentration of dATP? It will inhibit ribonucleotide diphosphate reductase. And this is exactly what happenes in a disease called Adenosine Deaminase Deficiency or the Boy in the Bubble. This boy has deficiency in adenosine deaminase, his immune system is unable to function, because there is no growth of immune cells, they are highly sensitive to dATP.

Treatment: Isolate the patient in a balloon structure, so it's called Bubble boy syndrome. Nowadays there is a cure for this disease; they insert by genetic engineering and genetic techniques a proper functional Adenosine gene in Bone marrow and it was a successful cure.

El 25tsar intsar . Y3tkm el 3afyeh . O sam7oni 3la el a5ta2 . O baha2 7ebebe msh 3arf shu a7kilk bs <3

DONE BY : AHMAD SHBOUL