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Synthesis and Degradation of Nucleotides Part 2: September 2 nd , 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.
Part 2: September 2nd, 2009
Champion CS Deivanayagam
Center for Biophysical Sciences and Engineering
University of Alabama at Birmingham
Birmingham, AL 35294-4400
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
Note that the numbering are slightly different and note where the glycosidic bonds are attached
1. What are the Committed steps that are unique in this synthesis cycle
2. What are the different feed back inhibition steps in this synthesis cycle
3. What steps can be utilized to develop inhibitors in this synthesis cycle
4. What are some of the diseases that are related to this synthesis cycle
In bacteria, six enzymes catalyze the reactions to form the pyrimidine ring
In mammals, these are encoded in three protein:
a. CPS-II, aspartatetranscarbomylase and dihydrorotate are in a 210 kDacytosolic polypeptide
b. DHO dehydrogenase is a separate enzyme associated with the outer surface of the inner
c. Orotatephosphoribosyltranferase and OMP carboxylase are encoded on a single cytosolic
polypeptide known as UMP synthase
The enzymatic activities are catalyzed by single polypeptide chains in mammals.
The advantages are:
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.
Transit time for movement from one active site to the next is shortened
Substrates are not diluted into the solvent phase
Chemically reactive intermediates are protected from decomposition into aqueous mileu
No pools of intermediates accumulate and
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.ColiATCase: Feebackinhibitied 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 Do Cells Form the Deoxyribonucleotides That Are Necessary for DNA Synthesis?
The enzyme system for dNDP formation consists of four proteins:
Two constitute the riboneuclotidereductase
Other two are Thioredoxin and Thioredoxinreductase
E. Coli RibonucleotideReductase Has Three Different Nucleotide-Binding Sites
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
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 thioredoxinreductase, mediates the NADPH-dependent reduction of thioredoxin.
Regulation of deoxynucleotide biosynthesis: the rationale for the various affinities displayed by the two nucleotide-binding regulatory sites on ribonucleotidereductase.
TrimericdCMPdeaminase. Each chain has a bound dCTP molecule (purple) and a Mg2+ ion (orange).
An alternative route to dUMP is provided by dCDP, which is dephosphorylated to dCMP and then deaminated by dCMPdeaminase.
It is allosterically activated by dCTP and feedback inhibited by dTTP.
Only dCTP does not interact with either regulatory sites on ribonucleotidereductase. Instead it acts upon dCMPdeaminase.
Synthesis of dTMP from dUMP is catalyzed by thymidylatesynthase
Thymidylatesynthasedimer. Each monomer has a bound folate analog (green) and dUMP (light blue).
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 thymidylatesynthase. The ternary complex is stable and prevents further enzyme turnover.
5-Fluorouracil is a thymine analog. It is converted to 5'-fluorouridylate by a PRPP-dependent phosphoribosyltransferase and passes through the reactions of dNTP synthesis, becoming 2'-deoxy-5-fluorouridylic acid, a potent inhibitor of dTMPsynthase. 5-Fluorocytosine is an antifungal drug, and 5-fluoroorotate is an anti-malarial drug.