الرحيم الرحمن الله بسم

Genetics lecture 2

The doctor said:

Ma3ako ya e5wan 30 secs etsakro el mobiles o raja2an kol mo7adara mn l mo7adarat l jay tkono m'3le8en l 5lawe kol el 5lawyat switched off, o b3den al raja2 3adam l do5ol wa 7amelen al 8ahwah wal shay wal mora6ebat!! Al mora6ebat mamnoo3a da5el l mo7adara!! ento msh

jayen t7daro cinema!! al maa2 msmoo7 da5l l mo7adara f86…

N8oool بسم الله الرحمن الرحيم

Now for This lecture, I will continue what I started last time, SO I will continue talking bout or the general objects for today's lecture:

» the degradation of purine and some clinical applications on that

» pyrimidine metabolism (biosynthesis and degradation) and some

clinical correlations on those aspects.

NOW, let us see this slide (#16), which represents part of the degradation of purine, so there is AMP and GMP, through this pathway they will end up with uric acid…

So>> taking a lot of purines will result in a lot of uric acid production and production of uric acid above normal concentration is considered as

disorder and that disorder in medicine called Gout.

***the general characteristic of Gout is: the hyperuricemia (the high concentrations of uric acid) that resulted from the degradation of purine nucleotides and when you have high concentration of uric acid in the

blood, it will precipitate in the joints as urate and that will cause a lot of pain…On slide # 17, there is some of the biochemical basis of Gout (the high concentration of uric acid in the blood), such as:

1- Elevated levels of phosphoryibosyl pyrophosphate (PRPP).

Q: what is the PRPP? Someone answered, but I didn't hear!! But the doctor said: this is a compound not an enzyme, so the PRPP is the precursor (general precursor) that we call upon metabolism or biosynthesis pathways that before the IMP and will give the different purines.∴ The accumulation or elevated levels of PRPP will cause high production of purines.

2- Increased biosynthesis of purine.

If you have an activated biosynthesis pathway of purine, you will end up with high uric acid because high synthesis means high degradation and then a lot of uric acid.

3- Apparent loss of feedback inhibition.

Q: What do we mean by this (apparent loss of feedback inhibition)? the doctor said: we are talking about the regulation of purine synthesis and as I mentioned last time that regulation is taking place by feedback inhibition of the end products, so if the site of regulation of the pathway for the biosynthesis or if we lost this inhibition, then there will be an accumulation of PRPP and as a result we will have a lot of purine and since we have a high amount of purine so the degradation product will be high (uric acid).

4- Partial deficiency of hypoxanthine –guanine phosphoryibosyl transferase (HGPRT).

Q: why Partial deficiency of HGPRT will cause gout? You can account for that if you imagine the salvage pathway………….

Suddenly some one kick the door and the doctor said "hada em3a9eb 3alana….!! (after a second men hada el 7aywan!! :O).

A Student answered then the doctor said: The salvage pathway converts PRPP with some purines to hypoxanthine and GMP or

AMP, if the HGPRT is partially deficient, what will be the result? An Accumulation of PRPP (the general precursor).

If PRPP is accumulated, that will go in the de novo and produce a lot of purine nucleotides that will be degraded in and produce a lot of uric acid.

5- Increased levels of uric acid exceed solubility of urate and leads to crystallization in joints and other areas.

6- When the Urate concentration is high it precipitates in the organs and leads to some disorders, BUT if it was totally deficient, it will not lead to goat but to X-linked genetic disease called Lesch-Nyhan syndrome.

In slide # 20: the Lesch-Nyhan syndrome and how patients of Lesch-Nyhan syndrome look like, some of the problems that may come up are mental problems and self humiliation, and the person starts to bind his organs (fingers, lips …..) and that bind will lead to death and that’s because of the accumulations of Uric Acid.

Now, in the Central Nervous System, the Salvage Pathway is predominating.

*** The complete deficiency or semi complete deficiency of HGPRT which is the principal enzyme for Salvage Pathway will lead to stop the synthesis and accumulation of PRPP and that will lead and produce some de novo and uric acid.

>>How uric acid in the brain will damage it? (Only Concentrations above the normal will lead to the Lesch-Nyhan syndrome, which is X linked inherited disease)…. (Slide # 19)

Q: how to treat Gout (or hyperuricemia)?

Answer: The drug is Allopurinol, and it will be active if it was converted to Alloxanthine, and Alloxanthine will inhibit the enzymes responsible for converting Hypoxanthine to xanthine and xanthine to Urate.

>>> Xanthine oxidase will convert allopurinol to its active form.

See Slide # 21.

*** Allopurinol is not harmful as a drug, but in the body it will be metabolized to its active form –Alloxanthine- and this will inhibit the xanthine oxidase itself and thus stop the formation of uric acid… this is called suicide inhibition of Allopurinol, because the if it was exposed to the xanthine oxidase it will be converted to its active form -Alloxanthine- which in turn will inhibit the synthesis of Uric acid.

?? One student asked a question but I couldn't hear the question but the Doctor answered: because the compound (Allopurinol) is not the proper substrate of these enzymes, it could be a substrate analogy but not the proper substrate, so these enzymes in the presence of allopurinol will be active, but when it is converted to Alloxanthine, that will inhibit these enzymes completely and thus uric acid production will stop..

??Another question which also I couldn’t hear but the doctor’s answer was: Allopurinol must be converted to Alloxanthine to be working on the treatment of Gout but Allopurinol itself can’t be harmful on the body, The treatment requires low concentrations of Uric acid in the body.

***Uric acid is good to the body in a normal physiological conditions but if it's go above the normal concentrations, it will be precipitated as urate in the joints the kidney and so many other organs then it will

damage those organs ,

SO the inhibition of high amount of production is negative to us but under normal conditions and control conditions, we want some uric acid because it acts in many aspects one of those is antioxidant (defensive

mechanism) .

•• In slide # 22, we can see that Uric acid and some other compounds

are good antioxidants such as bilirubin, glutathione and ascorbic acid.

••Another thing that it was found Uric acid concentration in

humans is a little higher than other primates (pigs, monkeys … etc).

••The high concentration of Uric acid was correlated to the increase

in the human being age, compared to other primates …. Do you know why we have longer ages than other primates? •Because Uric acid is an antioxidant and gets rid of free radicals that cause malignancies.

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

PYRIMIDINE METABOLISM

In slide # 23, you can see the main objectives that you have to understand after reading this lecture from our LOVELY BOOK!!

And anyone who doesn’t understand, just come and ask or go to the book and find the answer!! So you have to answer these questions and if you do it easily, there will be no problem for you from this course.

SO, look at the pyrimidine biosynthesis pathway on slide # 24 and from this slide you can obtain some differences between pyrimidine and purines biosynthesis.

Q: Any one wants to name some pyrimidine that we will talk about?

After asking six students who couldn’t know, finally Uracil comes up.

Q: what about pyrimidine nucleotides?

TMP, UMP and CMP.

**Glutamine is the amino acid responsible for the Nitrogen in the pyrimidines, so glutamine with ATP and HCO3 gives us the carbamoyl phosphate and glutamate.

The enzyme that catalyzes this is carbamoyl phosphate synthetase II.

While that for Urea is called carbamoyl phosphate synthetase I. ((be aware… ))

**Then the carbamoyl phosphate reacts with Aspartate and gives N-Carbamoylaspartate.

<<<Carbamoyl phosphate synthetase is a complete step and regulatory step in pyrimidine biosynthesis >>>

The next slide (# 25) shows us the differences between the two types of carbamoyl phosphate synthetase type 1 and type 2;

Type 1 is found in the liver and type 2 in all other tissues.

Q: why type 1 only in the liver but no other tissue?

Because of the Urea cycle which takes place in the liver.

Q: why type 2 is found in all the tissues?

Because of DNA synthesis and nucleic acid synthesis.

What concerns us is the parent compound as what in purines synthesis, >> the orotate that is comes to form carbamoyl phosphate and it starts the synthesis steps to give orotate, while some enzymatic oxidation - reduction reaction and the removal of the double bond and this will give us dihydrooratate. See slide # 26.

And then it will be oxidized and we will get orotic acid.

>>>>>>>Orotic acid is the parent compound that gives us the different pyrimidine

SO, as you can see, the orotate can give us:

- orotidylate by the orotate phosphoryibosyl transferase - The UTP & CTP by the orotidylate decarboxylase, we will get UMP

and the UMP will give us UTP & CTP… so this is what we concern of the UMP & CMP, then there will be double phosphorolated to give us the UTP & CTP. See both Slides # 26 & 27.

Quick note!! The CTP, CDP and the CMP are nucleotides.

??One student asked if we should memorize the name of the enzymes and the doctor said: NO.

??Another student asked: where is the dTMP in this pathway? And the doctor answered: the dTMP isn't emphasizing in this pathway, it has a

specific pathway we will mention it in a moment…..

Orotic aciduria: it is an inherited disease, if the enzymes that convert the orotic acid to UMP or CMP are deficient, there will be accumulations of orotic acid in the blood and this will cause an orotic aciduria

Some of the symptoms of this disease: large amounts of orotic acid in urine, retarded growth and severe anemia.

The treatment: injection of uridine or cytidine. See slide # 28.

So, what it is the purpose of slide # 31?

Before this, I want to answer a question; how dTMP is synthesized? It is synthesized by dUMP by the enzyme thymidylate synthetase (this enzyme is very important especially in your career).

This enzyme (thymidylate synthetase) requires: folate and tetrahydrofolate,, to convert dUMP to dTMP.

Q(A very very good question): from where we got this? Every thing we know is that we got UMP and UDP and UTP and somehow CTP but

dUMP has been seen for the 1st time, from where we got it??

The answer: the reduction of UMP…. ((Concentrate on it))

Q: How is it reduced?

By oxygen or removal of hydrogen >>> so it is an oxidation – reduction reaction.

SO, the enzyme that will work, converts the ribonucleotides to deoxy

ribonucleotides, and this enzyme is very very important, because the deficiency or inhibition of it will stop the whole life!! Because it will not have deoxy pyrimidines or purines to synthesize DNA. Of course if we have that enzyme deficient or inhibited.

That enzyme is called ribonucleotide diphosphate reductase.

And we will now talk more about it……

Q: Diphosphate reductase, could you correlate this with malignancy?? Could you correlate diphosphate reductase ribonucleotide or diphosphate reductase to malignancy? To cancer cells? How to correlate

that ??

The answer will be apparent later.……

This is not talk about the tissue, it only talk about the regulation and look at it!! It is highly complicated because it is very important and it must be under regulation because it is a crucial enzyme to synthesis,, why??

Because it converts ribonucleic acid to deoxyribonucleic phosphor DNA synthesis and gene synthesis and DNA replication and all these processes that we need, vital processes.

<The doctor wants us to think about the correlation of this enzyme and malignancy and cancer cells while he is explaining the regulation of the

enzyme>

Now, the substrates for this enzyme (in slide #32) are:

- CDP is to be converted to dCDP- UDP to be converted to dUDP, to dADP, to dGDP and then

another phosphorylation will convert them to deoxytriphosphate nucleotides

These are the positive allosteric effectors for this enzyme for each substrate….. Also in slide # 32.

SO, ATP is a positive effector for the diphosphate reductase, for each substrate; for the CDP and UDP substrates,, and dGTP is a positive effector, for ADP substrate and finally dTTP for GDP substrate, And these came by experiments.

These are the medical effectors (negative allosteric effectors) and if you notice, in all of these substrates for this enzyme, dATP is based, so high amounts of dATP is toxic.

** If you have high amounts of dATP, it will inhibit this enzyme as it allosteric effectors, and if this enzyme is inhibited, then there is no conversion of ribonucleotides to deoxyribonucleotides then there is no DNA replication, no gene expression.

So, high amounts of dATP is toxic to the cell and you must prevent high amounts of dATP above normal concentration, there is a disease, in which dATP is highly accumulated.

And this disease is called THE BUBBLE BOY SYNDROME.

Q: Who did read about bubble boy syndrome before 10 to 15 years?!!! So the purpose from the slide # 32 is to know that this enzyme has multi substrate, concentrates at the binding site, multi regulatory sites, for these different positive and negative allosteric sites and the importance of this is to convert (by reduction), the ribonucleotide to deoxyribonucleotide and why we need deoxyribonucleotide?? For DNA synthesis. If DNA synthesis stops then our life will stop.

Slide # 33, the doctor will leave it to us to read, but he said about it:

It's very easy, I've already mentioned about it, I don’t need it by explicitly, I’ve already mentioned that but not in clear! so I want you to read it and figure out if you understood the steps of synthesis of purines and pyrimidines, you must be able to differentiate between the 2 pathways...

“The Dr. asked if we have a problem in letting us read the slide by ourselves and said that he’s like to know if there is such a thing.”

Let us talk about the first one……….

Synthetic sequence:

In the case of purine, N- glycosidic bonds and then ring assembly and closure.

But in pyrimidines, ring assembly and closure and then N-glycosidic bonds.

?? Does that make sense to you?!! The things are opposite as one of the students said….SO>> firstly we have the sugar and then make the N- glycosidic bond in purines and then build on the N which is bonded to the sugar.

While in pyrimidines,, we make the carbamoyl phosphate and the orotate and then bring the sugar and the N- glycosidic bonds.

< So it’s easy to go over it by yourself >

The pyrimidine degradation:

This is the pyrimidine degradation (on slide # 35):

We have DNA and RNA and the result is acetyl CoA and methylmalonyl CoA and then goes to succinyl CoA.

DNA acetyl CoA

RNA methylmalonyl co!

((These are metabolic intermediates))

If the cell was degraded, then the DNA and RNA will be degraded and the enzymes that will degrade them are: DNase and RNase.

They will form TMP and then deoxythymidine OR UMP, dUMP, CMP and dCMP then dephosphorylation to produce deoxycytidine, cytidine, uridine and deoxyuridine.

BUT:

Q: Why we have cytidine and deoxycytidine? From where they come?

One from DNA and one from RNA, further dephosphorylation for them we come up with deoxyuridine and then they remove the sugar and return back to nitrogen bases (Thymine, uracil, cytosine) and then further metabolism to these nitrogen bases (thymine and uracil) till we reach to the succinyl coA and acetyl coA.

Q: What is the degradation product of purines?

Uric acid

Q: What are the degradation products of pyrimidines?

Acetyl CoA and succinyl CoA

NOW, you are familiar with purine nucleotides and pyrimidine nucleotides, you saw their importance in the 1st lecture; they are

involved in energy serving compound, they are important in DNA and RNA synthesis and regulation etc…

Before talking about this, I will talk about the correlation between ribonucleotide diphosphate reductase and cancer….

Q: Did you figure out any correlation or connection? Could you barn cancer cells by inhibiting this enzyme?

If we could inhibit the ribonucleotide diphosphate reductase by specific drugs or agents, you could stop the growth of cancer cells.

So this is the importance clinical correlation of the enzyme ribonucleotide reductase and cancer treatment,, SO if we look for inhibitors for that enzyme, you could control the growth of cancer cells, after treatment scientists trying their best to find drugs that will inhibit this enzyme for cancer treatment.

In slide # 38 some clinical signifances:

1) 6- Mercaptopurine : it may be involved in the biosynthesis of purine because it is a substrate analog, may involved as competitive inhibitor because it likes substrate, and inhibit some important enzymes for the synthesis of purines and this is exactly what happened.

So, 6- mercaptopurine is a drug that inhibits phosphoribosylpyrophosphate amidotransferase which is a committed step in purine biosynthesis; it inhibits the IMP DHASE and Adenylsuccinate synthase (synthesis of ATP).

2) 5-Fluorouracil (FUTP) and (FdUMP): this compound or these 2 compounds will inhibit the maturation of ribosomal RNA and messenger RNA. When we talk about the metabolism of those nucleic acids, you will see what do we mean by maturation biosynthesis and processing of these nucleic acids, so this compound FUTP will inhibit, and if you inhibit maturation and

processing of rRNA and mRNA, what are the consequences for that? Protein synthesis will stop and the cell will die.deoxy uridine monophosphate will inhibit thymidylate synthase (it is important in formation of deoxyTMP from dUMP and dUTP) so this enzyme is highly important because if you inhibit it, then you stop synthesis one of the components of DNA synthesis substrate which is the deoxyTTP so through deoxyUMP is an important drug or compound that will inhibit DNA synthesis by inhibiting synthesis of deoxyTMP.

3) Methotrexate : Inhibits dihydrofolate reductase.

Q (Important question): how Methotrexate will affect DNA and RNA synthesis?

>> So no need to explain it because you will do it………

4) Azaserine : Inhibits enzymes that utilize glutamine (gln) as N donors; it is gln antagonists.

Q: Is it good or bad?! It stops DNA synthesis and nucleotide synthesis!! So good or bad?!It is GOOD, because it could be used to fight cancer cells and viral genome, it could be good to fight bacterial genome that cause diseases to human being, BUT the question: if they find the genome of viral or bacterial or cancer cells, why they didn’t find the normal genome (the genome of normal cells) when they are used as drugs?!Now, suppose a person has a viral infection and that person is given a drug that will –for example- be gln antagonists like AZT or as a serine, the serine will fight the genome of the virus and kill it.

The question: does this compound -that fights the viral genome- fight the normal cells of the host?!! The doctor leaved this opened to US to stimulate our thinking!!!

5) 6-Diazo-5-oxo-1-norleucine : another compound which is Gln antagonists, it will inhibit the enzymes that utilize gln as N donors in purines and pyrimidines synthesis.

6) Hydroxyurea: Inhibits ribonucleotide reductase and it sometimes used as a drug to treat sickle cell disease.

The doctor want us to correlate the biochemical bases of using Hydroxyurea as drug to treat sickle cell disease and the mechanism of action, what is the relation of treatment of sickle cell disease by Hydroxyurea and the inhibition of ribonucleotide reductase by Hydroxyurea?

7) AZT (3-azido-3-deoxythymidine): antiviral-chain termination, does it make sense to you?! What do we mean by this?? This compound is antiviral and works by chain termination, but what do we mean by this? When the Virus grows, it must replicate the nucleic acid chain; either DNA or RNA, it must replicate, this means it is incorporated different nucleotides to make the new strand of DNA or new strand of RNA for the life cycle, this drug will be incorporated while the DNA strands is synthesized and the is analog to nucleotide, it will stop continuation of the DNA strand, so it will terminate the chain of DNA and RNA synthesis.

8) Acyclovir: is antiviral and it is purine analog and does the chain termination.

Slides # 39, 40 and 41 shows the structures of the compounds that are mentioned before, just to look at them not to memorize them.

Still to talk about bubble boy syndrome or adenosine deaminase deficiency in the next lecture……………………………..

GOOD LUCK

Done by: Yousef Odeh

………

Thanks for my friends Malik Hourani and Mohammad Al-

Okoor, and special thanks for the one who help me in this work to be as nice as possible, to go out for you all as it, and support me in every second while I do this lecture thanQ Very much

………