Purine Nucleotide Synthesis Regulation, Pyrimidine Synthesis, Pyrimidine Nulceotide Synthesis Regulation, Formation of Deoxyribonucleotides

Step 4

(4) Oxidation of dihydroorotate. -Dihydroorotate is irreversibly oxidized to orotate by dihydroorotate dehydrogenase. -Dihydroorotate dehydrogenase = eukaryotic enzyme; located on the outer surface of the inner mitochondrial membrane; powered by quinones (Q) -Inhibition of dihydroorotate dehydrogenase blocks pyrimidine synthesis in T lymphocytes, thereby attenuating the autoimmune disease rheumatoid arthritis

Step 4

(4) The radical-cation intermediate is reduced by the enzyme's redox-active sulfhydryl pair to yield a 3′-deoxynucleotide radical and a protein disulfide group

Step 5

(5) The 3' radical abstracts an H atom from the protein to yield the product deoxynucleoside diphosphate and restore the enzyme to its radical state.

Step 6

(6) Decarboxylation to form UMP. -Decarboxylation of OMP by OMP decarboxylase (ODCase) to form UMP. -The reaction requires no cofactors to help stabilize its putative carbanion intermediate.

Oxidized thioredoxin is reduced in a reaction by thioredoxin reductase

- FAD prosthetic group - Catalyzes the NADPH-mediated reduction of a substrate disulfide bond. -Read from right to left.

Step 3

(3) Ring closure to form dihydroorotate. -The third reaction of the pathway is an intramolecular condensation catalyzed by dihydroorotase to yield dihydroorotate.

Step 2

(2) Synthesis of carbamoyl aspartate. -Condensation of carbamoyl phosphate with aspartate to form carbamoyl aspartate is catalyzed by aspartate transcarbamoylase (ATCase). -This reaction proceeds without ATP hydrolysis because carbamoyl phosphate is already "activated."

Tetrahydrofolate Is Regenerated in Two Reactions

(1) DHF is reduced to THF by NADPH as catalyzed by dihydrofolate reductase (DHFR) 2) Serine hydroxymethyltransferase transfers the hydroxymethyl group of serine to THF yielding N5,N10-methylene-THF and glycine.

Step 1

(1) Ribonucleotide reductase's free radical (X·) abstracts an H atom from C3'of the substrate in the reaction's rate-determining step.

Step 1

(1) Synthesis of carbamoyl phosphate. -The first reaction of pyrimidine biosynthesis is the synthesis of carbamoyl phosphate from HCO3- and the amide nitrogen of glutamine by the cytosolic enzyme carbamoyl phosphate synthetase II. *This reaction consumes two molecules of ATP: One provides a phosphate group and the other energizes the reaction

Allosteric regulation of Class I ribonucleotide reductase

(1) The binding of ATP, dATP, dGTP, or dTTP to the specificity site induces the catalytically inactive R1 monomers to form a catalytically active dimer, R12. (2) The binding of dATP or ATP to the activity site causes the dimers to form tetramers, R14a, that slowly but reversibly change conformation to a catalytically inactive state, R14b. (3) The binding of ATP to the hexamerization site induces the tetramers to further aggregate to form catalytically active hexamers, R16, the enzyme's major active form. Monomers→Dimers→Tetramers→Hexamers -R1 is almost entirely in its tetrameric or hexameric forms due to [ATP] in a cell -ATP couples the overall rate of DNA synthesis to the cell's energy state.

Step 2/3

(2 and 3) Acid-catalyzed cleavage of the C2'—OH bond releases H2O to yield a radical-cation intermediate. The C3'-OH group's unshared electron pair stabilizes the C2' cation. This accounts for the radical's catalytic role.

5-Fluorodeoxyuridylate

-5-Fluorodeoxyuridylate (FdUMP) is an irreversible inhibitor of thymidylate synthase - In Step 3, however, the enzyme cannot abstract the F atom as F+ (F is the most electronegative element) so that the enzyme is frozen in an enzyme-FdUMP-THF ternary covalent complex.

Ribonucleotide Reductase Is Regulated by a Complex Feedback Network

-Feedback control - Maintaining the proper intracellular ratios of dNTPs is essential for normal growth -Excess is mutagenic Oligomeriztion of RNR is governed by the binding of nucleotide effectors to three independent allosteric sites on R1: (1) the specificity site, which binds ATP, dATP, dGTP, and dTTP; (2) the activity site, which binds ATP and dATP (3) the hexamerization site, which binds only ATP.

*Regulation of Pyrimidine Nulceotide Synthesis

-In bacteria, the pyrimidine biosynthetic pathway is primarily regulated at Reaction 2, the ATCase reaction. *In animals, carbamoyl phosphate synthetase II is the regulatory enzyme *CPSII is inhibited by UDP and UTP and activated by ATP and PRPP →In all organisms, the rate of OMP production varies with the availability of its precursor, PRPP.

Essential Protective Function

-Inability of Oxidized Ribonucleotide Reductase to Bind Substrate Serves an Essential Protective Function. - In the absence of substrate, the enzyme's free radical is stored in the interior of the R2 subunit. -If the substrate cannot properly react after accepting this free radical, (if the enzyme were in its oxidized state), the free radical could potentially destroy both the substrate and the enzyme. -An important role of the enzyme is to control the release of the radical's powerful oxidizing capability→preventing the binding of substrate while the enzyme is in its oxidized form

Orotic Aciduria

-Orotic aciduria, an inherited human disease, is characterized by the urinary excretion of large amounts of orotic acid, retarded growth, and severe anemia -Deficiency in the bifunctional enzyme catalyzing Reactions 5 (orotate phosphoribosyl transferase) and 6 (ODCase) of pyrimidine nucleotide biosynthesis. -Treatment: the administration of uridine and/or cytidine -The UMP formed through the phosphorylation of the nucleosides inhibits carbamoyl phosphate synthetase II; slows down the rate of orotic acid synthesis. -No other genetic deficiency in pyrimidine nucleotide biosynthesis is known in humans, presumably because such defects are lethal in utero.

Formation of Deoxyribonucleotides

-Ribonucleotide Reductase -DNA differs chemically from RNA in two major respects (1) Its nucleotides contain 2'-deoxyribose residues rather than ribose residues (2) it contains the base thymine (5-methyluracil) rather than uracil.

Structure of R22

-The Fe(III) complex interacts with Tyr 122 to form an unusual tyrosyl free radical -Free radical tyrosine important. Not attached to active site. Free radical prepares active site. Pull off hydrogen for the sugar. -The protein mediates electron transfer from this tyrosyl radical to another group that is closer to the substrate, probably the thiyl radical

Thioredoxin Reduces Ribonucleotide Reductase

-The final step in the ribonucleotide reductase catalytic cycle is reduction of the enzyme's newly formed disulfide bond to re-form its redox-active sulfhydryl pair -One of the enzyme's physiological reducing agents is thioredoxin

*Regulation of Purine Nucleotide Biosynthesis

-The pathways synthesizing IMP, ATP, and GTP are individually regulated in most cells -Ribose phosphate pyrophosphokinase (Reaction 1 of the IMP pathway) is inhibited by both ADP and GDP. -Amidophosphoribosyl transferase, the enzyme catalyzing the first committed step of the IMP pathway (Reaction 2) is likewise subject to feedback inhibition. -Amidophosphoribosyl transferase is allosterically stimulated by PRPP (feedforward activation).

Inhibition of Thymidylate Synthesis in Cancer Therapy

-dTMP synthesis is a critical process for rapidly proliferating cells -Interruption of dTMP synthesis can therefore kill those cells

Mechanism-based inhibitors (suicide substrates)

-enzyme inhibitors, such as FdUMP, that inactivate an enzyme only after undergoing part or all of the normal catalytic reaction - Extremely high specificity; among the most useful therapeutic agents -Inhibition of DHFR blocks dTMP synthesis as well as all other THF-dependent biological reactions, because the THF converted to DHF by the thymidylate synthase reaction cannot be regenerated. -Can stop Purine synthesis.

Synthesis of UTP from UMP

-is analogous to the synthesis of purine nucleoside triphosphates -CTP is formed by amination of UTP by CTP synthetase -In animals, the amino group is donated by glutamine -in bacteria, the amino group is supplied directly by ammonia.

Purines Can Be Salvaged

-the turnover of nucleic acids, particularly some types of RNA, releases adenine, guanine, and hypoxanthine and are reconverted to their corresponding nucleotides through salvage pathways. -Adenine phosphoribosyltransferase (APRT) mediates AMP formation using PRPP -Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyzes the analogous reaction for both hypoxanthine and guanine:

Ribonucleotide Reductase

-three classes of RNRs, which differ in their prosthetic groups, although they all replace the 2'-OH group of ribose with H via a free-radical mechanism. -Class I RNRs reduce ribonucleoside diphosphates (NDPs) to the corresponding deoxyribonucleoside diphosphates (dNDPs). -Class I RNRs contain an Fe prosthetic group and occur in most eukaryotes and aerobic prokaryotes. -E. coli ribonucleotide reductase mainly present in vitro as a heterotetramer that can be decomposed to two catalytically inactive homodimers, R12 and R22 -Each R1 subunit contains a substrate-binding site that includes several redox-active thiol groups. -The R1 subunits contain three effector-binding sites that control the enzyme's catalytic activity as well as its substrate specificity

Step 5

Dihydroorotate dehydrogenase

Lesch-Nyhan Syndrome

Results from HGPRT Deficiency. -Caused by a severe HGPRT deficiency -Results in excessive uric acid production -Symptoms include neurological abnormalities such as spasticity, mental retardation, and highly a -Lack of HGPRT activity leads to an accumulation of the PRPP →Increases synthesis of purine nucleotides and the formation of uric acid.

Pyrimidine Synthesis

The biosynthesis of pyrimidines is simpler than that of purines. -N1, C4, C5, and C6 of the pyrimidine ring are all derived from aspartic acid -C2 arises from HCO3- -N3 is contributed by glutamine. *UMP, which is also the precursor of CMP, is synthesized in a six-reaction pathway

dUMP Is Methylated to Form Thymine

The dTMP component of DNA is synthesized by methylation of dUMP. The dUMP is generated through the hydrolysis of dUTP by dUTP diphosphohydrolase (dUTPase) -dTMP, once it is formed, is phosphorylated to form dTTP

Thymidylate Synthase

Thymidylate (dTMP) is synthesized from dUMP by thymidylate synthase with N5,N10-methylenetetrahydrofolate (N5,N10-methylene-THF) as the methyl donor

Production of dNTPs

dNTPs Are Produced by Phosphorylation of dNDPs -The final step in the production of all dNTPs is the phosphorylation of the corresponding dNDPs