genetics lecture #2 , ahmad shboul

7/31/2019 Genetics Lecture #2 , Ahmad Shboul

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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 .


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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, metabolicpathways 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 guaninephosphoribosyl 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.


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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 novopathway, 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.


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There is another type called SECONDARY GOUT , and that resultsbecause 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 5-

phosphate 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 ofdeficiency 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 , X-

linked disease and it's a neurological disease and patients that have

complete deficiency of this enzyme , will have mental retardation and


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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.


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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 activatingAllopurinol 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 ATPplus 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 ) forurea cycle .


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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 westarted 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).


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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 toCTP.

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?


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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 butwhat 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 ribonucleotidediphosphate 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


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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 iswhat 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 .


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We want dTTP but this dTMP is easily converted by two successivephospholyrations 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, andyou 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.


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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


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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


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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:


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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.


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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 orthe 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.


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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 Adenosinegene in

Bone marrow and it was a successful cure.


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El 25tsar intsar . Y3tkm el 3afyeh .

O sam7oni 3la el a5ta2 .

O baha2 7ebebe msh 3arf shu a7kilk bs


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DONE BY :

AHMAD SHBOUL

genetics lecture #2 , ahmad shboul

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