الرحيم الرحمن الله بسم

Dr. nabeel basheer .. office :M2 L4, physiology department

The book that we use is Devline

This lecture will be on the metabolism of nucleotides ,it’s a completion of the metabolism coarse that deals with nucleotides

Since we are talking about genetics , specifically DNA ,genes and chromosomes , you must know how the building blocks of genes are formed and how they are degraded .

General objectives of this lecture and the coming one "nucleotide metabolism"

Why nucleotides are important in human metabolism??

There are some points you should think about ..

what is the relevance or the importance of nucleotides in human metabolism??

1-They are incorporated in DNA and RNA (building blocks )

DNA stands for deoxyribonucleic acid

RNA stands for ribonucleic acid

The difference between DNA and RNA is that DNA has no oxygen in the second carbon of ribose sugar

You know the base form of the nucleotide .. so knowing more about nucleotides and ribonucleotides enable you to understand the structure of DNA and RNA.

2 -Nucleotides are reservoirs for energy for metabolic reactions .

Adenine, guanine, and thymine are nitrogen basis but they are not nucleotides .

Adenosine and guanosine are nucleosides .

CTP, ATP and GTP are nucleotides

The difference between nucleoside and nucleotide ; is that nucleoside consists of ribose sugar and nitrogen base only but nucleotide consists of ribose sugar, nitrogen base in

addition to phosphate group .

So in studying metabolism you learned that ATP and GTP nucleotides are used as source of energy for biosyntheses, phosphorelation of other enzymes and for metabolites.

*Examples of metabolic reactions in which ATP is used in:

Na+/K+ pump.

Conversion of glucose to glucose-6 phosphate ( glucose was phosphorelated by ATP in the presence of hexokinase or glucokinase) which is an important process in glycolysis and carbohydrate metabolism.

3- These nucleotides are also used as carriers of activated intermediates

In glycogen biosyntheses(which is polysaccharides), glucose is used as building blocks in the form of UDP-glucose

-The series of the reactions in which glucose binds to the carrier:

glucose __phosphorelated glucose 6-phosphate __isomarizesed to glucose 1-phosphate UTP+ enzyme UDPG

So once glucose is linked to UDP, it is activated. It is proper substrate for glycogen synthase and it can be used for glycogen synthesis.

This is an example in which nucleotides can activate some intermediates to be used in metabolism as UDPG.

4- Nucleotides are structural compounds of essential co-factors as(CO-A, FAD, NAD+, and NADP+). (Nucleotide are parts of the structure of these components)

5- Regulators of metabolic enzymes (which means they will activate or inhibit other enzymes)

Glycogen phosphorelase is an important enzyme for glycogen degradation,the enzyme will be activated upon phosphorelation.

Phosphorelation is initiated by phosphorelase kinase or protein kinase-A in the presence of ATP which will activate it to glycogen phosphorelase .

Q/ What will happen for glycogen synthase when it becomes phosphorelated ?

IT will be inhibited

- So as we see here are regulators which means they will activate or inhibit other enzymes so glycogen phosphorelase will be activated by phosphorelaction in the presence of ATP while glycogen synthase will be inhibited by phosphorelation in the presence of ATP (which is a nucleotide)

So, they are important in different aspects in human metabolism in addition to being components of genes (a nucleic acids).

We will start talking about purin metabolism

- As you know nucleotides could be Purine components or pyrimidine components, so we will start talking about purin biosynthesis and purin degradation.

There are four Purines:

• Adenine = 6-amino purine • Guanine = 2-amino-6-oxy purine • Hypoxanthine = 6-oxy purine• Xanthine = 2,6-dioxy purine

The general formula of purin contains banzen ring (6 members ring) and emadazole ring (present in histidine).

You should know the numbering system in order to recognize these atoms.

Adenine has amino group on C-6 Guanine has amino group on C-2 and carbonyl group on C-6 Hypoxanthine instead of having an amino like in Adenine, it has a carbonyl group Xanthine has carbonyl group instead of amine group like in guanine

(you don’t want to memorize or to write the structure but if it was written for you then you must be able to recognize it)

The pyrimidines are:

• Uracil = 2,4-dioxy pyrimidine • Thymine = 2,4-dioxy-5-methyl pyrimidine • Cytosine = 2-oxy-4-amino pyrimidine • Orotic acid = 2,4-dioxy-6-carboxy pyrimidine

Orotic acid is a new one for you and will be seen in the metabolism of nucleotides

Nither Uracil is found in DNA nor Thymine is found in RNA

Thymine is specific for DNA and Uracil is specific for the RNA, BUT Cytosine is found in both.

You also have the numerating system. It starts from the nitrogen (the figure)

There are side chains that are attached to these groups in order to be differentiated from each others.

So you must be able to differentiate between cytosine and Uracil, For example the amino group on C4 in cytosine is replaced by carbonyl group in Uracil.

And must be able to differentiate between thiamine and Uracil, the side chain of methyl group which is on C5 on thymine but not present on uracil. [ you are going to see -when we are talking about the gene expression and DNA replication- what is the importance of having this methyl group in Uracil to change it from Uracil (which is not found in DNA) to thiamine

(which is found in DNA). it is crucial, dangers and important to having this methyl group in our genes . so without this methyl group we will not be able to live.

Before start talking about metabolic pathways of nucleotides there are some medical relevance of the nucleotides .

1. They are important in cancer treatment as you will see when we talk about it, I will mention some points that could be used for cancer treatment using those nucleotides.

An example of these : Hydroxyurea, anti-folates,florouracil (5-FU) and other compounds .

2. Some other nucleotides are also important in medicine .and some defects of nucleotides also will result in genetic or inherited diseases as in this disease Adenosine deaminase Deficiency (SCIDS) ,Lesch-Nyhan Syndrome and Hyperuricemia

All of these are defects or diseases because of the nucleotide metabolism pathway abnormality .

So during the pathway of nucleotide metabolism that will help you to understand the molecular basis of these diseases … you will be able to recognize which enzyme or which intermediate causes the disease related to the nucleotide metabolism .

Purine and pyrimidine nucleotide participate in many crucial cellular functions . nucleotides in the cell are supplied by de novo syntheses and by salvage of preformed nucleobases or nucleosides.

De novo syntheses pathway depends on starting from non cyclic compounds ( such as amino acids, CO2, and one carbon pool(such as tetrahydrofolate ) ) to synthesize cyclic compounds.

[In the figure] this is a general scheme to show you the pathway of syntheses of Purines and pyrimidine .

Q\What is the general overall picture of this graph??

A\it represents a complicated pathway or biosyntheses of Purines and pyrimidine but we will not go over all of these complicated things.

-The syntheses of Purines and pyrimidines in this pathway starts with the sugar (Ribose-5-phosphate) which we can get from hexomonophsphate shunt [ pentose shunt] in whish glucose is oxidized to gluconic acid and then to glucoronic acid and then to pentoses like ribelose and ribose -5-phosphate .

So that shunt is important to provide us with the precursor for the syntheses of nucleotides.

The steps of Purines syntheses in de novo pathway :

Ribose -5-phosphate will be phosphorelated by[ phosphoribosyl payrophosphate synthase (PRPP synthase )] to form phosphoribosyl payrophosphate (PRPP) and this compound will take one of the pathways to synthesize either Purines or pyrimidines.

*If you look at the structure of the Purines you will find that it is formed from carbon and nitrogen atoms which we will find out the source of each of them in general.

glutamine [ a polar amino acid ] will provide us with nitrogen.

* the difference between glutamine and glutamate(glutamic acid) that both of them are amino acids but glutamine is a polar amino acid and glutamate(glutamic acid) is an acidic amino acid that contains a carboxyl group .

glycine will provide the Purines with carbon skeleton and nitrogen. one carbon pool [it is a compound that donate a carbon atom like methyl , formyl

and tetrahydropholate (folic acid)which is used here to provide a methyl group as a one C-atoms components of Purines].

Glutamine will also be used as source of the nitrogen. carbon dioxide Co2 in the form of bicarbonate will be used as one of the carbon

sources aspartate (another amino acid) is used as a nitrogen source another molecule of carbon pool will be used as a source of carbon in purines.

*so you will see there is a mixture ; 2 glutamine , glycine , aspartate ,2 molecules of one carbon pool and HCO3 all of these will participate in synthesizing purines such as IMP(inozate monophosphate which is the parent compound of purines). IMP could go to AMP which is the parent compound of ATP and ADP ,, IMP could also go to GMP which is the parent compound of GTP and GDP .also IMP and GMP could be converted to urate and uric acid and form a metabolic diseases which are mentioned (hyper uresimia ).

The steps of pyrimidine syntheses in de novo pathway :

In pyrimidine syntheses we will use phosphoribosyl payrophosphate (PRPP) but we will not start with it

o It will start with aspartate and carbamyl phosphate to form orotate which in turn will react with PRPP to form UMP and then UMP will give UDP , UTP and it will also give dTMP(deoxi TMP) . UTP in turn will give CTP.

* we need dTMP and not TMP because TMP(the oxidized form) is not incorporated in RNA so we need only the deoxidized form (dTMP) which is incorporated only in the DNA and we also need deoxinucieotides which are incorporated in the DNA.

-so ,you have to remember the amino acids ,one carbon pool, co2 and ( the parent compound which gives AMPand GMP)in Purine syntheses pathway.

-also you have to remember the carbamyl phosphate and aspartate to give a parent compound to pyrimidine which is orotate which will react with PRPP to produce UMP which will give dTMP and CTP in series in pyrimidine syntheses pathway.

[figure] this figure is a skim shows the origin of each atom of Purines (where does it came from)

- You are not suppose to memorize the source of each atom( from where), but

you must know the idea that different amino acids and other compounds participate in the syntheses of Purine and pyrimidine nucleotides if these amino acids are not there or the folate folic acid is deficient then we will have some problems in growth ,synthesizing DNA and development .

- So… what you have to know is the medical relevance of these compounds, is the precursors that are important to be used in nucleotide biosyntheses

We said that for Purines biosyntheses we start with ribose-5-phosphate and it continues till it reaches to inozate -5-phosphate which will give AMP and GMP. [slide7]

The steps of the conversion of ribose-5-phoshat to inozate monophosphate:

(*it is not a matter of memorization of these reactions , I will not ask you about enzymes or intermediates but I want you to realize that it is not straight forward pathway to synthesis a single nucleotide to make compound )

(slide 9) ribose 5-phosphate will be phosphorelated by an enzymatic phosphorelation to producePRPP which will react with glutamine to form an amino sugar.

Glutamine is used as a sours of nitrogen, Which will react with PRPP by the action of (amino-phospho-ribozyl transpherase) , glutamate will be released , a glycosidic bond will be formed between the nitrogen and the sugar in the ribose amine and pyrophosphate(PPi) was removed (it is an important step).Q/ why do we have a pyrophosphate?For the activation of the ribose.

(slide 10) the ribose amine (phosphor-ribozyle-1-amine) will react with a molecule of glycine which will be attached to the amine.

In the next step, one carbon pool have been used (which means that we need one carbon moity either in the form of C=O or in the form of CH3 or formyl) [hear we use it as formyl]

next, another molecule of glutamine has been used to substitute the hydroxyl -group by an amine group.

Then this will be cyclized to form the emedazole ring which is a part of purin ring as we saw earlier.

In pyramidines you will not see a similar picture, you will see amino acids and carbon pools and other things….. will react with each other to form Orotate (as nitrogen base) and then the orotate will link to the sugar.

*In purins, we started from the sugar and everything was built on the sugar to form this ring, and as we saw already, the imidazole ring has been formed from these reactions…..

(you only need to recognize that the imidazole was formed by the reaction of ribose with glutamine (to form the nitrogen) and with glycine (to form the carbon) and one carbon pool ,and another gluramine (to give another nitrogen) and then the imidazole formed from the cyclization of this series of reactions ).

(just recognize what happened and appreciate the complicated processes that are taking place in our bodies).

Q\ How the imedazole is converted to inozate monophosphate to give a different purain nucleotides ??

1. 1-carbon pool(HCO3) will attach as COO‾ from its carbon atom to the ring 2. Another aspartate (aspartic acid) will bind from its amino group to the C (which

came from the carbon pool (in the previous step)3. Another carbon pool will substitute the carboxyl group that came from aspartate 4. Cyclization of the result of these processes will bind to the imedazole and Purine

ring will synthesized.

Generally Purine rings are composed of amino acids, carbon pools and CO2 .

*you have to recognize what amino acids and what carbon pools are used in order to form the Purine compounds that you must recognize it as inozate monophosphate.

Inozate (IMP) is the parent compound that gives AMP(which will give ATP and ADP) and GMP(which will give GTP and GDP) then this will be converted to deoxi form by specific enzymes which are very important.

- (Slide 12) to produce AMP from IMP … we react the IMP with aspirate in order to replace the carbonyl (oxi ) in the IMP by an amino which is taken from aspartate amino acid , the result compound is called Adenylosuccinate and you will end up with Inozate but instead of having an (oxi) carbonyl you have an amino group which is the AMP

So what is AMP?? It is an amino inozate monophosphate

- IMP (inozate monophosphate) will also end up with GMP

Inoszate in the form of IMP, it has an carboxyl group C=O,, it must be oxidized in order to be converted to guanilate:

1- Oxidation: addition of C=O (Carbonyl -group) to produce zanthilate (carboxi carbamil)

2- Zanthilate will react with glutamine in order to change the Carbonyl to amino (which is taken form glutamine) and glutamate will be released and thus the guanilate mono-phospho (GMP) will be produced. -so guanilate mono-phosphate (GMP) is simply IMP but in addition there is some oxidation in to oxo-carbonyl and then amination to end up with GMP*you must be able, if you were given these two structures (slide 12) AMP and GMP, to differentiate between them by looking at the functional groups(Carbonyl and amino groups)

Once you have AMP and GMP you will be able to have ATP, ADP, GTP, and GDP and you will also be able to have the deoxi form of these nucleotides

-Slide 14: control and regulation:

- It is important to have control and regulation for AMP and GMP pathways of synthesis, and it is not acceptable to be working all the time, if that happened then there will be over production of nucleotides and deoxi-nucleotides and that will be toxic to the cells so there must be a regulatory mechanism and controls to switch the mechanism of synthesis on/off

As you can see on the graph, these are the sights of regulation

-If you have a lot of AMP,, by feedback inhibition then AMP will inhibit the step which convert the inozate monophosphate (IMP) to adenolate succinate that will give AMP, so excess AMP will control its self and stop the step that produce it.

-GMP, if the cell has excess amount of GMP synthesized, so GMP will stop this.

The 1st and the 2nd steps (slide 14) will be inhibited by IMP, AMP, and GMP by negative feedback and allosteric inhibition that will control these enzymes.

So these are the sights that are subjected to regulation by the products their selves (IMP, GMP, and AMP).

That will control these sights inhibited enzymessuch as amylotrasferaze enzymes .

The other pathway of purin biosynthesis which is the salvage pathway:

In this pathway, we didn’t start from scratch (amino acids and carbon pools) but we start from ribose-5-phosphate and cyclic compounds of Purines which can be obtained from the diet or from other already synthesized compounds.

-slide 15 explains this salvage pathway

Starting with PRPP (which is a purin) that is already found in the cell or obtained from the diet to synthesize purin nucleotides.

There are two enzymes which are important in the salvage pathway

1- Hypoxanthin-guanin-phospho-ribozyl-transferase (HGPR) that can use the already made hypoxanthin in the cell and attach it to PRPP to make the proper nucleotides.

2- Adinin-phosphoribozyl-transferaze (APRT) that will use adenine derivatives which have adenine cyclic groups from the already found in cells and attach them to PRPP in order to form the proper adinolate nucleotide.

It is easier that the De-novo, and it is rich in many tissues especially in brain tissues which seems that it depends on the salvage pathway for the synthesis of Purine nucleotides

-The deficiency of this enzyme will lead to a metabolic diseases such as Lesch-Nyhan syndrome. This syndrome will cause an accumulation of of deoxy-ATP that will be toxic to the brain cell and that will form a lot of uric acid (hyperurecemia) and that will cause … for those people who have deficiency of this enzyme.

If there is a partial deficiency then there is no big deal but if there are a complete deficiency then that will cause lesch-nyhan syndrome which will cause mental retardation because of the hyperureciemia which is toxic to the brain cells.

Slide 16 is important: there are AMP and GMP ,and when degraded by a specific enzyme then AMP is converted into IMP which in turn will be converted to hypoxanthin which will be converted to xanthin

GMP converts directly (so AMP and GMP when metabolized they are converted into what is so called xanthin)

xanthin will be oxidized by an enzyme called xanthin oxidase and … convert xanthin into uric acid

If there is excess of AMP and GMP then there will be excess of uric acid and that will cause hyper urecemia which will cause gout which results from precipitation of uric acid in the joints and form a painful gout.

So gout is a metabolic disease which results from a deficiency in purine nucleotide metabolism, if there is no control or regulation of the synthesis of AMP and GMP then accumulation of them will be converted into xanthin and xanthin is oxidized to uric acid causing hyper urecimian which will cause goute .

Forgive me if there are any mistakes

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Done by: Asma k. Abu-Salah