Chapter 18 - Nucleotide Biosynthesis

• precursor of DNA and RNA

• ATP and GTP

• components of co-factors (NAD, FAD, coenzyme A)

• biosynthetic intermediated

• second messengers (cAMP and cGMP)

what are nucleotides important in

second messengers

intracellular signaling molecules released by the cell to trigger physiological change

first messengers

extracellular substances that include peptide hormones

amino acids, ribose 5-phosphate, CO2, and NH3

what can nucleotides be synthesized de novo from

nucleobases

what can nucleotides be salvaged from

most lack de novo biosynthesis and rely exclusively on salvage

in what way do most parasite synthesize nucleotides

ribose

in de novo biosynthesis of nucleotides, the bases are synthesized while attached to

glutamate

what amino acid provides most amino groups in nucleotide de novo synthesis

glycine

what amino acid is the precursor for purines

aspartate

what amino acid is the precursor for pyrimidines

kept low so cell must continuously synthesize them which may limit rates of transcription and replication

why are nucleotide pools kept low and what might it affect

RNA has OH group at 2’ position while DNA has an H

difference between RNA and DNA nucleotides

• aspartate provide a nitrogen

• CO2 provided a carbon

• formate provided 2 carbons

• glutamine provided 2 amide nitrogens

• glycine provided N-C=C

origin of ring atoms in purines

isotopic experiments with 14C- or 15N- labeled precursors

how was it determined the origin of ring atoms in purines

reaction of PRPP with glutamate

what does synthesis of purines begin with

purine ring builds up following addition of 3 carbons from glycine

what occurs after PRPP and glutamate react during purine synthesis

inosinate / inosinic acid / inosine monophosphate (IMP)

what is the first intermediate with full purine ring

IMP + aspartate + GTP (adenylosuccinate synthetase) → adenylosuccinate (adenylosuccinate lyase) → AMP

how is AMP synthesized from IMP

IMP (IMP dehydrogenase) → XMP (XMP-glutamine amidotransferase) → GMP

how is GMP synthesized from IMP

1. end products IMP, GMP, and AMP inhibit glutamine-PRPP amidotransferase enzyme

2. excess GMP inhibits IMP dehydrogenase which blocks formation of XMP from IMP

3. GTP limits conversion of IMP to AMP and ATP limits conversion of IMP to GMP

4. ADP and GDP inhibit PRPP synthesis

4 major mechanisms of feedback inhibition in regulation of purine biosynthesis in E. coli

aspartate, PRPP, and carbamoyl phosphate

what are pyrimidines made from

first making the pyrimidine ring and then attaching it to ribose 5-phosphate

how does pyrimidine synthesis process first and then after

reaction between aspartate and N-carbamoylphosphate which is catalyzed by aspartate transcarbamoylase (ATCase)

what is the first committed step in the synthesis of pyrimidines

allosteric regulation

regulation by binding other than active site

ATP accelerates it while CTP inhibits it

how do ATP and CTP impact ATCase activity

2’ C-OH bond is directly reduced to 2’-H bond without activating the carbon → catalyzed by ribonucleotide reductase

how are ribonucleotides converted to deoxyribonucleotides (basic)

2 H atoms are donated by NADPH and carried by proteins thioredoxin or glutaredoxin

mechanism by which 2’C-OH bond of ribonucleotide is reduced to 2’-H bond of deoxyribonucleotide

NADPH donates H atoms to GSSG converting it to 2GSH which donates them to glutaredoxin which then donates them to ribonucleotide reductase

what is involved in the transfer of H atoms from NADPH to glutaredoxin

H atoms are donated from NADPH to FAD generating FADH2 which transfers them to thioredoxin which transfers them to ribonucleotide reductase

what is involved in the transfer of H atoms from NADPH to thioredoxin

one type affects activity and the other type affects substrate specificity

2 types of regulatory sites that ribonucleotide reductase has

ATP activates and dATP inhibits

ribonucleotide reductase regulatory site affecting activity

• when ATP or dATP is bound, reduction of UDP and CDP is favored

• when dTTP or dGTP is bound, reduction of GDP or ADP is stimulated respectively

ribonucleotide reductase regulatory site affecting substrate specificity

oligomerization of the enzyme → a2b2 becomes ring-shaped a4b4 (inactive)

what occurs to ribonucleotide reductase when dATP binds

UDP (ribonucleotide reductase) → dUDP (nucleotide diphosphate kinase) → dUTP (dUTPase) → dUMP (thymidylate synthase) → dTMP

steps to get dTMP from UDP

deamination

how is dUTP made from dCTP

adds a methyl group from tetrahydrofolate (specifically the

N⁵,N¹⁰-methylenetetrahydrofolate)

how does thymidylate synthase convert dUMP to dTMP

thymidylate

what does folic acid deficiency lead to reduced synthesis of

causes uracil to be incorporated into DNA and repair mechanisms remove it by creating strand breaks that affect DNA structure and function (associated with defects)

result of reduced thymidylate synthesis

hypoxanthine via deamination and hydrolysis reactions

in the catabolism of purines what is adenosine first broken down to

xanthine via hydrolysis and deamination reactions

in the catabolism of purines what is guanosine first broken down to

uric acid by xanthine oxidase

during purine catabolism what are xanthin and hypoxanthine oxidized into and via what enzyme

urea via the urea cycle than as uric acid from purine degradation

what to primates more commonly excrete nitrogen as

• urate is oxidized into a 5-hydroxyisourate by urate oxidase

• hydrolysis and the subsequent decarboxylation of it leads to allantoin

steps of degradation of urate to allantoin

most mammals - allantoin

humans - urate

what form do most mammals vs humans excrete nitrogen in from the degradation of purines

uric acid → allantoin → allantoate → urea → ammonium cation

what can uric acid from degradation of purines be converted into

NH₄⁺ then urea and can produce intermediates of citric acid cycle

what does the catabolism of pyrimidines lead to

thymine → methylmalonylsemialdehyde → succinyl-CoA

what molecules in thymine degraded down into and what can that molecules be further degraded to

they are recycled by salvage pathways

what happens to purine and pyrimidine bases released in metabolism

adenine reacts with PRPP to the form AMP using the enzyme adenosine phosphoribosyltransferase

example of how adenine base is reused

Lesch-Nyhan syndrome

what disorder does lack of hypoxanthine-guanine phosphoribosyl transferase lead to

gout → painful joints

what can excess uric acid cause

• avoidance of purine-rich foods or fructose

• allopurinol → xanthine oxidase inhibitor

how can gout be treated (2 ways)

• allopurinol is similar in structure to hypoxanthine (normal substrate of xanthine oxidase)

• at the active site of enzyme, allopurinol is converted to oxypurinol which is a strong competitive inhibitor that remains tightly bound to the reduced form of the enzyme

how does allopurinol work to treat gout

glutamine analogs that inhibit glutamine amidotransferase (interfere with many amino acid and nucleotide biosynthetic pathways)

what are azaserine and acivicin and what do they inhibit

it’s converted into FdUMP which inhibits thymidylate synthase

what does fluorouracil inhibit

dihydrofolate reductase

what do methotrexate and aminopterin inhibit