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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.

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slide1

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

slide2

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

slide3

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

slide4

What do you need to learn from this lecture ?

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

slide5

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
slide13

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

mitochondrial membrane

c. Orotatephosphoribosyltranferase and OMP carboxylase are encoded on a single cytosolic

polypeptide known as UMP synthase

slide14

The advantages of multifunctional enzymes:

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

slide15

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

slide16

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
slide17

How Do Cells Form the Deoxyribonucleotides That Are Necessary for DNA Synthesis?

  • In most organism NDP’s are the substrates fordeoxyribonucleotide formation.
  • Reduction at 2'-position commits nucleotides to DNA synthesis
  • Replacement of 2'-OH with hydride is catalyzed by ribonucleotidereductase
  • Three classes of ribonucleotidereductases differ in their mechanisms of free radical generation

The enzyme system for dNDP formation consists of four proteins:

Two constitute the riboneuclotidereductase

Other two are Thioredoxin and Thioredoxinreductase

slide18

E. Coli RibonucleotideReductase Has Three Different Nucleotide-Binding Sites

  • An 22-type enzyme - subunits R1 (86 kD) and R2 (43.5 kD)
  • R1has 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 thioredoxinreductase deliver reducing equivalents
slide19

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

slide20

RibonucleotideReductase 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 thioredoxinreductase, mediates the NADPH-dependent reduction of thioredoxin.

slide22

RibonucleotideReductase is Regulated by Nucleotide Binding

Regulation of deoxynucleotide biosynthesis: the rationale for the various affinities displayed by the two nucleotide-binding regulatory sites on ribonucleotidereductase.

slide24

How Are Thymine Nucleotides Synthesized?

  • Cells have no requirement for free thymine ribonucleotides and do not synthesize them
  • dUDP anddCDP 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 dCMPdeaminase yielding dUMP.
slide25

dCMPDeaminase Provides an Alternative Route to dUMP

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.

slide26

Synthesis of dTMP from dUMP is catalyzed by thymidylatesynthase

Thymidylatesynthasedimer. Each monomer has a bound folate analog (green) and dUMP (light blue).

  • ThymidylatesynthasemethylatesdUMP 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.
slide27

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 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.