Biochemistry Ch 25 – Nucleotide Biosynthesis

Bases
– A, G, T, U,
C
Purines – A, G Pyrimidines – C, U, T
Nucleoside – base coupled to 5 C ribose or deoxyribose sugar via β-glycosidic linkage
Ribonucleosides – adenosine, guanosine, uridine, cytidine
Deoxyribonucleosides – deoxyadenosine, deoxyguanosine, thymidine, deoxycytidine
Nucleotide – nucleoside with phosphate attached
Ribonucleotides – adenylate, guanylate, uridylate, cytidylate
Deoxyribonucleotides – deoxyadenylate, deoxyguanylate, thymidylate, deoxycytidylate

Nucleotide roles
 Precursors of RNA & DNA
 ATP – adenine nucleotide used as universal energy currency
 GTP – guanine nucleotide, serves as energy source for more select processes
 UDP-glucose – nucleotide derivative used in biosyntheses such as glycogen synthesis
 Cyclic nucleotides – act as second messengers in signal transduction (e.g., cyclic AMP)

General pathway of de novo synthesis

 Pyrimidines – bases are built as individual molecules, then they are linked to activated ribose
 Purines – bases are assembled while bound to activated ribose
 Deoxyribonucleotides are made by reduction of ribonucleotides
 Thymine (a deoxy base) is made by modification of deoxy uracil nucleotide (dUMP)

Salvage pathway – a base is reattached to an activated ribose

de novo pathway – base itself is synthesized from simpler starting materials (amino acids, CO 2), requiring ATP
hydrolysis and an activated ribose
5-Phosphoribosyl-1-pyrophosphate (PRPP) – a form of ribose activated to accept nucleotide bases

- Bases are added at C1, the pyrophosphate at C1 is

Pyrimidine de novo synthesis what makes it active bc it is a good leaving group and
 Ring is synthesized first, then it iscan
attached
be furthertohydrolyzed
PRPP to make rxn irreversible
 Step 1 – Carbamoyl phosphate (remember- Phosphates arestart
it from added at C5cycle)
of urea
o Synthesis of carbamoyl phosphate from bicarbonate and ammonia in a multistep process
catalyzed by carbamoyl phosphate synthetase (CPS), requiring 2 ATPs:

o Source of NH3 is primarily glutamine side chain, not free ammonia

o CPS binds glutamine at one end and hydrolyzes it to get NH 3 which is funneled through the
interior of CPS to the two ATPase sites, “substrate channeling”, remaining in interior of
enzyme and avoiding contact of reactive species with H 2O; carbamoyl-P released at other end

 Step 2 – Orotate
o Carbamoyl phosphate reacts with aspartate, catalyzed by aspartate transcarbamoylase
o Intermediates are cyclized and oxidized with NAD to yield orotate
  Step 3 – Orotidylate
o Orotate couples to PRPP to form orotidylate, a pyrimidine nucleotide (OMP)
o The enzyme that catalyzes this addition is pyrimidine phosphoribosyltransferase

 Step 4 – Uridylate (uracil nucleotide)

o Orotidylate (OMP) is converted to uridylate (UMP) by decarboxylation
o UMP is used for RNA synthesis and is precursor of cytidine and thymidine

 UMP can be converted as needed to UDP & UTP

o Nucleoside monophosphates are converted into diphosphates by specific nucleoside
monophosphate kinases that utilize ATP as the phosphoryl-group donor:
UMP + ATP  UDP + ADP via UMP kinase
o Nucleoside diphosphates are converted into triphosphates (or the reverse) by a general
nucleoside diphosphate kinase:
UDP + XTP  UTP + XDP (swaps 3rd P from one to another)

 Conversion of uracil to cytosine

o Only occurs after UMP is converted to UTP, as explained above, because the enzyme only
works on UTP (takes UTP  CTP)
o Uracil carbonyl group is replaced by amino group (from glutamine), a reaction that requires
ATP hydrolysis

Pyrimidine synthesis overview:

(1) Synthesis of carbamoyl phosphate from
bicarbonate and Gln-derived ammonia
(2) Formation of orotate ring from
carbamoyl phosphate & aspartate
(3) Coupling to activated ribose PRPP to
form orotidylate OMP
(4) Decarboxylation of OMP to form
uritidylate (UMP)
(5) Phosphotransfer to UMP to form UTP
(6) Amination of UTP to form CTP
* conversion to dNTPs discussed later *

Purine de novo synthesis

 Purines are synthesized piece by piece while attached to the ribose framework
 Step 1 – Amination of ribose
o Initial committed step: displacement of pyrophosphate from PRPP by glutamine-derived
ammonia, resulting in 5-phosphoribosyl-1-amine
PPi

NH3
 Next several steps
o The next six steps are analogous reactions: displacement of phosphates by amines
o Each step consists of activation of a carbon-bound oxygen atom by phosphorylation, followed
by displacement of the phosphoryl group by ammonia or an amine group as a nucleophile:

o We just need to know some of the donors in the reactions: Gly, Gln, Asp, formyl-THF, CO2
o Know that fumarate is released, and both rings are closed, resulting in inosinate (IMP)
IMP is a purine used as “mother molecule” to produce AMP and GMP:
 Inosinate yields adenylate and guanylate
o Amination of IMP (via Asp-derived amino group) with release of fumarate results in
adenylate, requiring GTP as the phosphate donor
o Oxidation of IMP (with NAD+) and amination (via Gln-derived amino group) results in
guanylate, requiring ATP as the phosphate donor

o Note that the synthesis of AMP requires GTP, whereas the synthesis of GMP requires ATP.
This reciprocal use of nucleotides by the two pathways creates an important regulatory
opportunity:
 Low GTP means production of adenylate will be reduced, increasing the production of
guanylate
 But adenylate is the precursor for ATP, so as less adenylate is produced that means less
ATP is now available to make GMP
 Low ATP means production of guanylate will be reduced, increasing the production of
adenylate
 But guanylate is the precursor for GTP, so as less guanylate is produced that means less
GTP is available to make AMP
 And so the regulatory cycle continues…
 Purine synthesis overview:
(1) Amination of PRPP via displacement of
its PPi with amine group
(2) Multiple phosphorylations via ATP
which activate CO groups for
displacement with amine group. Sources
of atoms include Gly, Gln, Asp, THF-
formyls, CO2
(3) Resulting IMP is aminated to yield
AMP and GMP
(4) As with the pyrimidines, the AMP and
GMP are easily converted to ATP and GTP
via phosphotransfer by specific and
general kinases

Synthesis of deoxyribonucleotides
 So far we have produced purines and pyrimidines in the form of ribonucleotides (UTP, CTP, ATP, GTP)
 The precursors of DNA, deoxyribonucleotides, are formed by the reduction of ribonucleotides,
specifically, the reduction of the 2’-hydroxyl group on the ribose to a hydrogen
 The reductant used is NADPH, the enzyme that catalyzes the reduction is ribonucleotide reductase –
the same enzyme will bind and reduce all four ribonucleotides (binds them as diphosphates, NDPs)
 The initial products of this reaction are therefore dNDPs (dUDP, dCDP, dADP, dGDP)
 Thus, further processing is required to convert the uridylate to thymidylate
o DNA contains thymine, the methylated analog of uracil
o This methylation is catalyzed by thmyidylate synthase which requires deoxyuridylate (dUMP)
as its substrate (so the initial product, dUDP, will transfer its P to form dUMP)
o Methylation of dUMP (donor is methylene-THF) results in thymidylate (TMP) & dihydrofolate
o Dihydrofolate can be reduced with NADPH via dihydrofolate reductase to regenerate the THF

Thymidylate synthesis blocked by anticancer drugs

 Rapidly dividing cells require an abundant supply of thymidylate for synthesis of DNA
 Inhibition of TMP synthesis machinery is thus a target for cancer drug therapy, because most of your
cells won’t be affected by decreased TMP production, but rapidly dividing cancer cells will
 Flurouracil
o anticancer drug that blocks TMP synthesis via inhibition of thymidylate synthase
o Forms fluorodeoxyuridylate (F-dUMP) which irreversibly binds thymidylate synthase and is
not released from the enzyme
o This is an example of “suicide inhibition” in which the enzyme is trapped in a form that cannot
proceed down the reaction pathway
 Dihydrofolate analogs
o Synthesis of TMP can also be blocked by inhibiting the regeneration of THF
o Analogs of dihydrofolate (aminopterin & methotrexate) are competitive inhibitors of
dihydrofolate reductase (the enzyme that is used to regenerate THF from dihydrofolate)
o Inhibition of dihydrofolate reductase thus inhibits the regeneration of THF, which inhibits the
production of TMP (because TMP synthesis requires methylation of dUMP by means of
methylene-THF)
Regulation of nucleotide biosynthesis
 Nucleotide biosynthesis is regulated by feedback inhibition in a manner similar to regulation of aa
biosynthesis: the regulatory pathways ensure that the various nucleotides are produced in the
required quantities and ratios
 Pyrimidine regulation
o The step in pyrimidine synthesis where aspartate and carbamoyl phosphate form the orotate
ring is catalyzed by aspartate transcarbamoylase
o Aspartate transcarbamoylase is inhibited by CTP, the final product of pyrimidine synthesis
o Aspartate transcarbamoylase is stimulated by ATP
 Purine regulation
o Committed step in purine nucleotide synthesis is the amination of PRPP, which is feedback-
inhibited by IMP, AMP, and GMP
o The two paths of inosinate, to AMP or GMP, are also feedback inhibited, so GMP inhibits the
conversion of inosinate to GMP; AMP inhibits the conversion of inosinate to AMP
o Also remember the inosinate pathways are regulated by GTP and ATP (already discussed –
because GTP is required for AMP synthesis, and ATP is required for GMP synthesis)

Regulation of deoxyribonucleotides
 The synthesis of dNTPs is controlled by the regulation of ribonucleotide reductase (the enzyme that
catalyzes the reduction of ribonucleotides to yield deoxyribonucleotides)
 Each subunit in the ribonucleotide reductase enzyme has two allosteric binding sites, one of which
regulates overall activity, the other regulates substrate specificity
o Overall catalytic activity is diminished by the binding of dATP, which is a signal of
overabundance of all the deoxyribonucleotides. The binding of ATP reverses this feedback
inhibition.

o Specificity for nucleotides is determined by each dNTP binding to the specificity allosteric site
on the enzyme, each stimulating the binding of the other (non-self) nucleotides. This complex
pattern of regulation supplies the appropriate balance of the four deoxyribonucleotides
needed for the synthesis of DNA.

Nucleotide metabolic problems

 Turnover of nucleotides
o Dephosphorylation of nucleotides by nucleotidases
o Cleavage to bases and ribose-1-P by nucleoside phosphorylases
 Ribose-1-P is isomerized by phosphoribomutase to yield ribose-5-P, a substrate in the
synthesis of PRPP
 Some of the bases are reused to form nucleotides via salvage pathways, others are degraded to
products that are excreted
 Deficiency in enzymes can disrupt these breakdown pathways, leading to various pathological
conditions
o Failure of deamination of adenosine is associated with severe combined immunodeficiency
(SCID- the bubble boy disease) – an ineffective immune system due to loss of T-cell activity
o Breakdown of purines results in uric acid which is converted to urate to be excreted in urine.
Higher than usual purine catabolism results in elevated levels of urate in the bloodstream,
which crystallize and resulting the joint disease gout
o Disruption of the salvage pathway via genetic mutation of guanine phosphoribosyltransferase
(the enzyme that couples guanine to PRPP) results Lesch-Nyhan syndrome (compulsive self-
destructive behavior) and mental deficiencies. The elevated levels of PRPP lead to an increase
in the rate of purine biosynthesis by the de novo pathway, and accordingly, an overproduction
of urate.
o Folic acid deficiency can lead to neural tube defects (spina bifida). More folate derivates may
be needed for the synthesis of DNA precursors when cell division is frequent and substantial
amounts of DNA must be synthesized (growth & development during pregnancy) .