Chapter 26- Nucleotide Metabolism

How can nucleotides by synthesized?

De novo pathways= pathways in which nucleobases are assembled from scratch
Salvage pathways = pathways in which performed bases are connected and recovered and reconnected to a ribose unit

Difference between pyrimidine and purines for de novo synthesis

• For pyrimidines, the framework for the base is assembled first and then attached to ribose

• For purines, the base is synthesized piece by piece directly onto a ribose-based structure

Pyrimidine de novo biosynthesis

*know what comes in (bicarbonate aspratate and ammonia) know what attaches, and what comes out

• The pyrimidine ring is assembled from bicarbonate, ammonia, and aspartate

  • Glutamine often serves as an ammonia donor

    

• Bicarbonate and other oxygenated carbon compounds are activated by phosphorylation

  • Carbamoyl phosphate is synthesized from bicarbonate and ammonia in a multistep process

    • requires 2 ATP

    • catalyzed by carbamoyl phosphate synthetase II

• Carbamoyl phosphate synthetase II includes sites for 3 reactions

  • ATP grasp fold= structure that surrounds ATP and holds it in an orientation suitable for nucleophilic attack at the γ phosphoryl group

  • Carbamoyl phosphate synthetase II contains 2 ATP grasp domains

  • Side chain of Glutamine can be hydrolyzed to generate ammonia

    • Carbamoyl phosphate synthetase II contains three active sites: 

      1. Glutamine hydrolysis site

      2. Bicarbonate phosphorylation site

      3. Carbamic acid phosphorylation site

• Intermediates move between sites by means of substrate channeling

  • ensures intermediates are captures with no loss caused by diffusion

  • protects alterable intermediates from hydrolysis

• The pyrimidine ring is completed and coupled to ribose

  • Carbamoylaspartate is metabolized to orotate

  • In mammals, enzymes that form orotate are part of a single large polypeptide chain called CAD (for carbamoyl phosphate synthase, aspartate transcarbamoylase and dihydroorotase).

• Orotidylate is decarboxylated to form UMP

  • Uridylate (UMP)= a major pyrimidine nucleotide that is a precursor to RNA

  • Orotidylate is decarboxylated to form UMP

    • catalyzed by orotidylate decarboxylase

• Nucleotide Mono-, Di-, and Triphosphates are interconvertible

  • Nucleoside monophosphates are converted into disphosphates by specific kinases

    • Ex. UMP is phosphorylated to UDP by UMP kinase

  • Nucleoside diphosphate kinases catalyzes the interconversion of di- and triphosphates 

    • Catalyzed by nucleoside diphosphate kinase 

• CTP is formed by amination of UTP

  • CTP is formed from UTP by the replacement of a carbonyl group with an amino group

    • catalyzed by cytidine triphosphate synthetase & requires ATP

    • uses glutamine as an amino group source

How is ribose 5-phosphate activated to become part of the nucleotide?

• Through the formation of  5-phosphoribosyl-1-pyrophosphate (PRPP)

  • PRPP= a form of ribose activated to accept nucleobases

  • Synthesized by 5-phosphoribosyl-1-pyrophosphate synthetase

    • uses 1 ATP

How are pyrimidines such as thymidine recovered in the salvage pathway?

• Salvage pathways recycle pyrimidine bases

  • Thymine released from DNA degradation, is salvaged in two steps:

    • Step 1: thymidine phosphorylase converts thymine into thymidine

    • Step 2: thymidine kinase converts thymidine into a nucleotide

Purine de novo biosynthesis

• Purine bases can be synthesized from glycine, aspartate, and other components

  • Purine bases are assembled already attached to the ribose ring

  • The committed step in purine biosynthesis forms 5’-phosphoribosyl-1-amine from PRPP and glutamine

    • The amine is in the β configuration

    • Catalyzed by glutamine phosphoribosyl amidotransferase 

• The purine ring is assembled by successive steps of activation by phosphorylation followed by displacement

  • Nine additional steps are required to assemble the ring

  • Each step consists of activation of an oxygen by phosphorylation followed by displacement of phosphoryl group by ammonia or an amine group acting as a nucleophile

  • ATP provides the driving force to favor biosynthesis 

• Inosinate can be converted to AMP and GMP

  

Purine salvage pathway

• Salvage pathways economize intracellular resource consumption

  • Adenine phosphoribosyltransferase catalyzes the formation of adenylate (AMP) from adenine and PRPP

• Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) catalyzes the formation of guanylate (GMP) and inosinate (IMP) from Guanine and PRPP

Deoxyribonucleotide formation

• Deoxyribonucleotides are synthesized by the reduction of ribonucleotides

  • Ribonucleotide reductase = catalyzes the replacement of a hydrogen atom for the 2’-hydroxyl group on the ribose parts of a ribonucleoside diphosphate, yielding a deoxyribonucleotide

    • Uses NADPH as a reductant 

    • Catalyzes the reduction reaction for all four ribonucleotides

Purine Nucleotide biosynthesis regulation

• The synthesis of purine nucleotides is controlled by feedback inhibition at several sites

  • Glutamine phosphoribosyl amidotransferase catalyzes the committed step in purine synthesis (conversion of PRPP into phosphoribosylamine)

    • Inhibited by AMP and GMP

  • Inosinate is the branch point in the synthesis of AMP and GMP

    • AMP inhibited the conversion of inosinate into adenylosuccinate, its immediate precursor 

    • GMP inhibits the conversion of inosinate into xanthylate, its immediate precursor

  • GTP is a substrate in the synthesis of AMP, whereas ATP is a substrate in the synthesis of GMP

    • Tends to balance the synthesis of adenine and guanine ribonucleotides 

    • PRPP synthetase is also highly regulated even though it is not the committed step in purine synthesis 

      

Primidine Nucleotide biosynthesis regulation

• Key steps in nucleotides biosynthesis are regulated by feedback inhibition

  • Aspartate transcarbamoylase (ATCase) regulates the biosynthesis of pyrimidine nucleotides

  • ATCase is inhibited by CTP, an example of feedback inhibition

  • ATCase is stimulated by ATP

Ribonucleotide reductase regulation

• The synthesis of deoxyribonucleotides is controlled by the regulation of ribonucleotide reductase 

  • Each E. coli ribonucleotide reductase R1 subunit contains two allosteric sites:

    • A site controlling the overall activity of the enzyme 

    • A site regulating substrate specificity

  • Binding of dATP inhibit enzyme activity, an effect reversed by ATP

• Regulation of substrate specificity

  • dATP or ATP binding to substrate-specificity control site enhances the reduction of UDP and CDP

  • Thymidine triphosphate (TTP) binding promotes the reduction of GDP and inhibits the further reduction of pyrimidine ribonucleotides

  • An increase in the level of dGTP stimulates the reduction of ATP to dATP

• Ribonucleotides Reductase is highly regulated to balance the pool of deoxyribonucleotides 

  

For purines how do you make PRPP?

You need to have ribose 5-phosphate that gets phosphorylated using ATP to AMP