BCHM 4116 Chapter 26

What are the purines?

adenine and guanine

What are the pyrimidines?

cytosine, uracil, thymine

Why are purines and pyrimidines important?

-activated precursors of DNA and RNA

-structural moieties of coenzymes (NADH, FAD)

-critical elements of energy metabolism (ATP, GTP)

-secondary messengers (cAMP, cGMP)

-metabolic allosteric regulators

Why is de novo synthesis of purine and pyrimidine bases important?

because dietary uptake of purine and pyrimidine bases is minimal

Because rapidly proliferating cells synthesize more DNA and RNA what does that say about the number of nucleotides produced?

there is a larger number of nucleotides produced

Where does the purine biosynthesis pathway occur?

occurs in the liver

How many steps is the purine synthesis pathway?

11 steps

How many ATPs are used for every purine synthesized?

6

What are some precursors that donate components to produce purine nucleotides?

glycine, ribose-5-phosphate, glutamine, aspartate, etc.

What is the goal of purine synthesis and why?

inosine monophosphate, which is the initial product of the 11-step purine nucleotide pathway

What is the starting point of the purine de novo pathway?

-synthesis of PRPP

-conversion of ribose-5-phosphate to PRPP via PRPP synthetase (ATP to AMP)

5’-phosphoribosyl-1-pyrophosphate (PRPP)

the activated form of ribose that purines are built on

What regulates PRPP synthetase?

-activated by inorganic phosphate

-inhibited by purine nucleotides (ADP and GDP)

What does end-product inhibition of PRPP synthetase mean?

it is a crucial regulatory mechanism in cellular metabolism where the final products of purine and pyrimidine nucleotide pathways bind to the PRPP synthetase enzyme to inhibit its activity; form of a negative feedback loop

T/F: synthesis of PRPP is the first committed step of de novo purine synthesis

FALSE

What is the first step of the de novo purine synthesis pathway?

-synthesis of 5-phosphoribosyl 1-amine

-conversion of PRPP to 5-phosphoribosyl 1-amine via glutamine phosphoribosyl amidotransferase

What is the mechanism behind the conversion of PRPP to 5-phosphoribosyl 1-amine?

the amide group of glutamine replaces the pyrophosphate group attached to the C1 of PRPP

What inhibits glutamine phosphoribosyl amidotransferase in the purine synthesis pathway?

AMP and GMP, another example of end-product inhibition!

Why is the production of 5-phosphoribosyl 1-amine important?

it produces nitrogen 9 of the purine ring; this nitrogen is now in the B-configuration and will be important for future B-glycosidic bonds

What is step 2 of the de novo purine synthesis pathway?

-glycine is added to the growing precursor

-conversion of 5-phosphoribosyl 1-amine to glycinamide ribosyl 5-phosphate via phosphoribosylglycinamide synthetase

What is the importance of glycine for the synthesis of glycinamine ribosyl 5-phosphate?

glycine provides carbons 4 and 5 and nitrogen 7 of the purine ring

What does the synthesis of glycinamide ribosyl 5-phosphate require?

energy, specifically in the form of ATP

Where does carbon 8 in IMP come from?

N10-formyl-FH4

Where does nitrogen 3 in IMP come from?

glutamine

Where does carbon 6 in IMP come from?

CO2

Where does Nitrogen 1 in IMP come from?

aspartate

Where does Carbon 2 in IMP come from?

N10-formyl-FH4

How many high energy bonds of ATP are required to synthesize IMP?

6

What is the base of IMP?

it has a hypoxanthine base which is joined by an N-glycosidic bond from N-9

What is the precursor to adenosine monophosphate and guanosine monophosphate?

IMP

How many steps are the pathways for adenosine and guanosine?

2 steps which require either ATP or GTP

What energy source is needed to synthesize adenosine monophosphate?

GTP

What energy source is needed to synthesize guanosine monophosphate?

ATP

T/F: the AMP and GMP pathways also have end product inhibition

TRUE; the first reaction of each pathway (after IMP) is inhibited by the end product of that pathway

Once AMP and GMP are made, what happens next?

they can then be phosphorylated to their di and tri phosphate forms which can be used for RNA synthesis

What do GDP and ADP inhibit?

PRPP synthetase, preventing the synthesis of PRPP

What do GMP, AMP, and each of their di and tri phosphate analogs inhibit?

glutamine phosphoribosyl amidotransferase (part of 1st committed step)

What do AMP and GMP inhibit?

the first step in their synthesis from IMP

What are the products of de novo purine biosynthesis?

the nucleotide monophosphates adenosine monophosphate and guanosine monophosphate which are then converted to ATP and GTP via additional phosphorylation reactions

What converts AMP to ADP and GMP to GDP?

base-specific ATP-dependent kinases (both use ATP as energy source)

What is responsible for the conversion of ADP to ATP?

oxidative phosphorylation

Nucleoside Diphosphate Kinase

enzyme that catalyzes the reaction for ATP serving as a phosphoryl donor for synthesis of other nucleoside triphosphates from their corresponding diphosphates

What is the goal of the purine salvage pathway?

to save ATP

T/F: pyrimidines have a salvage pathway

FALSE

How can nucleotides be resynthesized in the purine salvage pathway?

by reacting a free base with PRPP, known as a salvage reaction

What enzyme carries out a salvage reaction?

phosphoribosyltransferases

Hypoxanthine-Guanine Phosphoribosyltransferase

the enzyme responsible for the resynthesis of guanine/hypoxanthine in a purine salvage pathway

Adenine Phosphoribosyl-Transferase

the enzyme responsible for the resynthesis of adenine in a purine salvage pathway

What is PRPP used for in the purine salvage pathway?

it is used as a source of ribose-5-phosphate

5’-Nucleotidase

the enzyme responsible for converting nucleotides to nucleosides in the purine salvage pathway

Nucleoside Phosphorylase

the enzyme responsible for converting free bases from nucleosides

What is the only NUCLEOSIDE that can be salvaged and how?

adenosine, it is phosphorylated directly back to AMP by adenosine kinase

Lesch-Nyhan Syndrome

-x-linked recessive disorder associated with a complete deficiency of hypoxanthine-guanine phosphoribosyl transferase

-inability to salvage hypoxanthine or guanine

In patients with Lesch-Nyhan Syndrome, what does the lack of salvage pathway do to IMP, PRPP, and GMP levels?

increased PRPP levels and decreased IMP and GMP levels

In patients with Lesch-Nyhan syndrome, what happens to de novo purine synthesis?

the first committed step in purine synthesis will now have excess substrate and decreased inhibitors leading to increase of de novo purine synthesis

In patients with Lesch-Nyhan syndrome, what happens to purine and uric acid levels?

-the combination of decreased purine salvage and increased purine synthesis results in increased degradation of purines

-excessive amounts of uric acid are produced (end product of purine degradation)

In patients with Lesch-Nyhan syndrome, what is a common symptom?

-mental retardation (severe form)

-prone to chewing off their own fingers and other acts of self-mutilation

Where are purines degraded?

purines synthesized de novo will be degraded in the liver. purines ingested from food will be degraded in the small intestine

What is the first step of the purine degradation pathway?

AMP is deaminated to IMP via AMP deaminase

What is the second step of the purine degradation pathway?

IMP and GMP are dephosphorylated to their nucleoside forms via 5’-nucleotidase

Purine Nucleoside Phosphorylase

enzyme responsible for the conversion of inosine and guanosine into their respective purine bases, hypoxanthine and guanine, via cleaving of ribose from the base in purine degradation

Guanase

enzyme responsible for deamination of guanine to produce xanthine in purine degradation

Xanthine Oxidase

enzyme responsible for oxidizing hypoxanthine to produce xanthine and then oxidizing xanthine to uric acid in purine degradation pathway

Gout

-disorder indicated by high levels of uric acid in the blood as a result of either the overproduction or inadequate excretion of uric acid

-uric acid will ionize to urate which is insoluble and will crystallize in tissues, causing an inflammatory response (arthritis)

Allopurinol

structural analog of hypoxanthine which will inhibit uric acid synthesis which is used in patients who are overproducers of uric acid; used as a treatment for gout

What is the mechanism of allopurinol?

it is converted to oxypurinol which inhibits xanthine oxidase, resulting in accumulation of hypoxanthine and xanthine which are more soluble than uric acid and therefore less likely to initiate an inflammatory response

What is the goal of pyrimidine synthesis?

production of UMP

What is different about the pyrimidine ring synthesis in comparison to purine synthesis?

-unlike the synthesis of the purine ring which is constructed on a pre-existing ribose-5-phosphate, the pyrimidine ring is synthesized before being attached to the ribose-5-phosphate donated from PRPP

-the ring is built first and PRPP comes in at the end

What is the origin of the atoms in the pyrimidine ring?

aspartate and carbamoyl phosphate, derived from CO2 and glutamine

How many steps are in the pyrimidine synthesis pathway?

7

What is the initial product of the pyrimidine nucleotide pathway?

UMP

What is the first step of the pyrimidine synthesis pathway?

conversion of glutamine to carbamoyl phosphate via carbamoyl phosphate synthetase II

What are the regulators of CPS II?

-inhibited by uridine triphosphate

-activated by PRPP

Compare and Contrast CPS I vs CPS II

-CPS I is involved in the urea cycle, uses NH4+ as its nitrogen source, is located in the mitochondria and is activated by N-acetylglutamate (no inhibitors)

-CPS II is involved in the synthesis of pyrimidines, uses glutamine as its nitrogen source, is located in the cytosol and is activated by PRPP (inhibited by UTP)

What is step 2 of pyrimidine biosynthesis?

asp is added to carbamoyl phosphate via aspartate transcarbamoylase

What is step 3 of pyrimidine biosynthesis?

the addition of asp causes the molecule to produce a closed ring which is oxidized to form orotic acid (orotate)

What is step 4 of the pyrimidine biosynthesis pathway?

transfer of ribose-5-phosphate from PRPP to orotate to form orotidine 5’-phosphate via orotate phosphoribosyltransferase

What is step 5 of the pyrimidine biosynthesis pathway?

decarboxylation of OMP via orotidine 5’-phosphate decarboxylase to form UMP; IMPORTANT REGULATORY STEP

What is step 6 of the pyrimidine biosynthesis pathway?

UMP is phosphorylated to UDP and UTP

What is step 7 of the pyrimidine biosynthesis pathway?

CTP and TTP can be derived from UTP for RNA and DNA synthesis

Hereditary Orotic Aciduria

-disorder in which orotate phosphoribosyltransferase and orotidine 5’-phosphate decarboylase are defective, stopping UMP production

-treatment is oral administration of uridine which can be converted to UMP

Why would a patient with Hereditary Orotic Aciduria not grow?

if the enzymes that contribute to UMP production are blocked, there will be an increase in orotic acid in urine and decrease in UMP, leading to CTP or TTP production which will mean RNA/DNA synthesis will be decreased, body won’t be able to grow

What is the first step in the UTP and CTP synthesis pathway?

UDP is formed from UMP via ATP-dependent enzyme, nucleoside monophosphate kinase

What is the second step in the UTP and CTP synthesis pathway?

UTP is then formed by nucleoside diphosphate kinase

How is CTP produced after UTP is formed?

CTP is produced by amination of UTP via CTP synthetase which uses glutamine as the nitrogen source

What is the importance of CTP?

CTP can be dephosphorylated to CDP which can be used as a substrate for ribonucleotide reductase

What can the dCDP product be used for?

can be phosphorylated to dCTP for DNA synthesis or dephosphorylated to dCMP that can be deaminated to dUMP

Thymidylate Synthase

enzyme responsible for conversion of dUMP to dTMP using N5,N10-methylene FH4 as its methyl group source

5-fluorouracil

-forms FdUMP which inhibits thymidylate synthase

-used as a treatment for colon cancer

Why would 5-FU be used for cancer treatment?

because the blocking of purine or pyrimidine synthesis is an effective way to inhibit cell proliferation

What is the regulated step in pyrimidine synthesis?

CPSII step

What inhibits CPSII?

UDP and UTP

What activates CPSII?

PRPP and ATP

What is the relationship between pyrimidine concentration and CPSII activation?

as pyrimidines decrease in concentration (indicated by UTP levels), CPSII is activated and pyrimidines are synthesized

T/F: pyrimidine free bases can be salvaged

FALSE, pyrimidine bases can be recycled from nucleosides which can be phosphorylated to nucleotides but free pyrimidine bases cannot be salvaged

Why are pyrimidine salvage pathways less clinically significant?

because they are highly soluble

What is the two-step route of pyrimidine salvage?

-pyrimidine nucleoside phosphorylase will convert pyrimidine bases to respective nucleosides

-more specific nucleoside kinases will react with nucleosides to form nucleotides

What is the difference between the purine ring and pyrimidine ring?

unlike the purine ring which is not cleaved in humans, the pyrimidine ring is opened and degraded to highly soluble products

What are the steps of pyrimidine degradation?

-pyrimidine nucleotides are hydrolyzed to their nucleosides + Pi

-nucleosides are cleaved to produce ribose-1-phosphate and free pyrimidine bases (C, U, T)

-cytosine is deaminated to form uracil which can then be converted to CO2, NH3, and B-alanine

-excreted in urine or converted to CO2, H2O and NH3 (forms urea)

What is the difference between pyrimidine and purine catabolism?

catabolism of pyrimidine bases does not cause problems unlike catabolism of purine bases which can lead to gout; normal cellular metabolites are produced