Transcript Biosynthesis of Nucleotides 2 - University of Alabama at Birmingham

Synthesis and Degradation of
Nucleotides
Part 2: September 2nd, 2009
Champion CS Deivanayagam
Center for Biophysical Sciences and Engineering
University of Alabama at Birmingham
Birmingham, AL 35294-4400
Recollection’s from yesterday’s lecture
1. The purine ring is built on a ribose-5-P foundation through 11 steps to get IMP
2. GMP and AMP are derived from IMP
Today’s lecture will concentrate on Pyrimidine synthesis and catabolism
Gylocosidic bond
Gylocosidic bond
Note that the numbering are slightly different and note where the glycosidic bonds are attached
What do you need to learn from this lecture ?
1.
2.
3.
4.
What are the Committed steps that are unique in this synthesis cycle
What are the different feed back inhibition steps in this synthesis cycle
What steps can be utilized to develop inhibitors in this synthesis cycle
What are some of the diseases that are related to this synthesis cycle
De novo pyrimidine synthesis:
 In contrast to purines, pyrimidines are not synthesized as nucleotides
 Rather, the pyrimidine ring is completed before a ribose-5-P is added
 Carbamoyl-phosphate and aspartate are the precursors of the six atoms of the
pyrimidine ring
 Mammals have two enzymes for carbamoyl phosphate synthesis – carbamoyl
phosphate for pyrimidine synthesis is formed by carbamoyl phosphate synthetase II
(CPS-II), a cytosolic enzyme
DHO’s immediate e- acceptor is quinone
In bacteria, six enzymes catalyze the reactions to form the pyrimidine ring
In mammals, these are encoded in three protein:
a. CPS-II, aspartate transcarbomylase and dihydrorotate are in a 210 kDa cytosolic polypeptide
b. DHO dehydrogenase is a separate enzyme associated with the outer surface of the inner
mitochondrial membrane
c. Orotate phosphoribosyltranferase and OMP carboxylase are encoded on a single cytosolic
polypeptide known as UMP synthase
The advantages of multifunctional enzymes:
The enzymatic activities are catalyzed by single polypeptide chains in mammals.
The advantages are:
1. The product of one reaction in a pathway is the substrate for the next, and the
product remains bound and are channeled directly to the next active site rather than
disassociated into the surrounding medium for diffusion to the next active site.
2. Transit time for movement from one active site to the next is shortened
3. Substrates are not diluted into the solvent phase
4. Chemically reactive intermediates are protected from decomposition into aqueous
mileu
5. No pools of intermediates accumulate and
6. Intermediates are shielded from interactions with other enzymes that might
metabolize them
A comparison of the regulatory circuits that control pyrimidine
synthesis in E. coli and animals.
E.Coli ATCase: Feeback inhibitied by the end product CTP
ATP is an allosteric regulator
CTP and ATP compete for a common allosteric site.
CPS II in mammals: UDP and UTP are feed back inhibitors
PPRP and ATP are allosteric regulators
How Are Pyrimidines Degraded?
• In some organisms, free pyrimidines are
salvaged and recycled to form nucleotides via
phosphoribosyltransferase reactions
• In humans, however, pyrimidines are recycled
from nucleosides, but free pyrimidine bases are
not salvaged
• Catabolism of cytosine and uracil yields alanine, ammonium ion, and CO2
• Catabolism of thymine yields -aminoisobutyric
acid, ammonium ion, and CO2
How Do Cells Form the Deoxyribonucleotides That Are Necessary for
DNA Synthesis?
• In most organism NDP’s are the
substrates for deoxyribonucleotide
formation.
• Reduction at 2'-position commits
nucleotides to DNA synthesis
• Replacement of 2'-OH with hydride is
catalyzed by ribonucleotide reductase
• Three classes of ribonucleotide
reductases differ in their mechanisms
of free radical generation
The enzyme system for dNDP formation consists of four proteins:
Two constitute the riboneuclotide reductase
Other two are Thioredoxin and Thioredoxin reductase
E. Coli Ribonucleotide Reductase Has Three Different NucleotideBinding Sites
An 22-type enzyme - subunits R1 (86 kD) and R2 (43.5 kD)
R1 has two regulatory sites, a specificity site and an overall activity site
Activity depends on Cys439, Cys225, and Cys462 on R1 and on Tyr122 on R2
Cys439 removes 3'-H, and dehydration follows, with disulfide formation between
Cys225 and Cys462
• The net result is hydride transfer to C-2'
• Thioredoxin and thioredoxin reductase deliver reducing equivalents
•
•
•
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R1 homodimer carries two type of regulatory
sites in addition to the catalytic site
Catalytic site binds substrates: ADP, CDP,
GDP and UDP
One regulatory site binds:
ATP, dATP, dGTP or dTTP
Depending on which one of the nucleotides
is bound there determines which NDP is bound at the catalytic site
Other regulatory site binds: ATP (the activator) or dATP (the negative effector)
Overall activity site that determines whether the enzyme is active or inactive
The 2 Fe atoms within the single active site formed by the R2 homodimers generate the free
radical required for ribonucleotide reduction on a specific R2 residue, Tyr 122.
This in turn generates the thiyl free radical (Cys-S·) on Cys439.
Cys439-S· initiates ribonucleotide reduction by abstracting the 3’ H from the ribose ring of
the nucleoside diphosphate substrate and form s a free radical on C-3’.
Subsequent dehydration forms the deoxyribonucleotide product
Ribonucleotide Reductase Uses a Free Radical Mechanism
Cys residues undergo reversible oxidation-reduction between (-S-S-) and (-SH-SH-)
In their reduced form serve as electron donors to regenerate the reactive –SH pair in the active site
The sulfhydryls of thioredoxin reductase, mediates the NADPH-dependent reduction of thioredoxin.
Ribonucleotide Reductase is Regulated by Nucleotide Binding
Regulation of deoxynucleotide biosynthesis: the rationale for the various affinities
displayed by the two nucleotide-binding regulatory sites on ribonucleotide
reductase.
How Are Thymine Nucleotides Synthesized?
• Cells have no requirement for free thymine ribonucleotides and do not
synthesize them
• dUDP and dCDP lead to the formation of dUMP the immediate precursor for
dTMP synthesis
• Interestingly, formation of dUMP from dUDP passes through dUTP, which is then
cleaved by dUTPase, a pyrophosphatase that removes Ppi from dUTP.
• The action of dUTPase prevents dUTP from serving as a substrate in DNA
synthesis.
• An alternative route to dUMP formation starts with dCDP, which is
dephosphorylated to dCMP, and then deaminated by dCMP deaminase yielding
dUMP.
dCMP Deaminase Provides an Alternative Route to dUMP
An alternative route to dUMP is provided by dCDP, which is Trimeric dCMP deaminase. Each
dephosphorylated to dCMP and then deaminated by dCMP chain has a bound dCTP molecule
(purple) and a Mg2+ ion (orange).
deaminase.
It is allosterically activated by dCTP and feedback inhibited
by dTTP.
Only dCTP does not interact with either regulatory sites on
ribonucleotide reductase. Instead it acts upon dCMP
deaminase.
Synthesis of dTMP from dUMP is catalyzed by thymidylate synthase
•
Thymidylate synthase methylates dUMP at
5-position to make dTMP
•
N5,N10-methylene THF is 1-C donor
•
Once again folate derivatives are used as
inhibitors to disrupt DNA synthesis similar
to the purine synthesis.
Thymidylate synthase dimer.
Each monomer has a bound
folate analog (green) and dUMP
(light blue).
Fluoro-Substituted Analogs as Therapeutic Agents
Carbon-fluorine bonds are extremely rare in nature, and
fluorine is not common in nature.
Moreover, F is
electronegative and relatively unreactive.
Thus fluoro-substituted agents are often potentially useful drug
candidates. Shown here is the effect of 5-fluoro substitution
on the mechanism of action of thymidylate synthase. The
ternary complex is stable and prevents further enzyme
turnover.
5-Fluorouracil is a thymine analog. It is
converted to 5'-fluorouridylate by a PRPPdependent phosphoribosyltransferase and
passes through the reactions of dNTP
synthesis,
becoming
2'-deoxy-5fluorouridylic acid, a potent inhibitor of
dTMP synthase. 5-Fluorocytosine is an
antifungal drug, and 5-fluoroorotate is an
anti-malarial drug.