Amino Acid and Nucleotide Biosynthesis and One-Carbon Reactions

Nitrogen Fixation and Initial Ammonia Incorporation

Atmospheric Nitrogen Overview: Approximately $80\%$ of the Earth’s atmosphere consists of $N_2$ . This is an inert gas characterized by a very high bond energy of $225\,kcal\,mol^{-1}$ . This gas serves as the ultimate source of all nitrogen found in proteins, nucleotides, and various prosthetic groups within the biosphere.
Biological Nitrogen Fixation: - Nitrogen-fixing bacteria, such as Rhizobium, utilize $ATP$ and the enzyme nitrogenase to convert $N_2$ into ammonia ( $NH_3$ ). - Nitrogen-reducing bacteria fix approximately $1 \times 10^{11}\,kg\,N_2\,yr^{-1}$ .
Glutamate Dehydrogenase (GluDH): - This enzyme is the primary mechanism for fixing all $NH_4^+$ into proteins. - Reaction Direction: It is reversible; however, in the reductive direction, it scavenges free ammonia to produce amino acids. - The First Step: GluDH catalyzes the first step for all amino acid biosynthesis because glutamate eventually donates its $NH_4^+$ to all other amino acids. - Cofactors: It uses either $NAD(P)H + H^+$ as electron donors. - Stereospecificity: GluDH is stereospecific, producing exclusively L-glutamate. - Reaction Equation: $\alpha \text{-ketoglutarate} + NH_4^+ + NAD(P)H + H^+ \rightleftharpoons \text{L-glutamate} + NAD(P)^+ + H_2O$ .

Amino Acid Biosynthesis Families and Carbon Skeletons

Six Biosynthetic Families: In bacteria and plants, amino acid synthesis is organized into six families based on common precursors. These pathways generally preserve the number of "contiguous" carbons.
Carbon Skeleton Origins: The precursors for amino acids are derived from three central metabolic pathways: - Citric Acid Cycle (CAC): Provides skeletons for the $\alpha \text{-ketoglutarate}$ family ( $5C$ : Glutamate, Glutamine, Proline, Arginine) and the Oxaloacetate family ( $4C$ : Aspartate, Asparagine, Methionine, Threonine, Isoleucine, Lysine). - Glycolysis: Provides skeletons for the $3 \text{-phosphoglycerate}$ family ( $3C$ : Serine, Cysteine, Glycine), the Pyruvate family ( $3C$ : Alanine, Valine, Leucine), and the Phosphoenolpyruvate family. - Pentose Phosphate Pathway (PPP): Provides Erythrose $4 \text{-phosphate}$ (used with PEP for aromatic amino acids) and Ribose $5 \text{-phosphate}$ (used for Histidine).

Synthesis of Specific Amino Acids (3- and 5-Carbon Families)

Glutamine Synthesis: - Enzyme: Glutamine synthetase. - Reaction: $ATP + NH_3 + \text{Glutamate} \rightarrow \text{Glutamine} + ADP + P_i$ . - Affinity: This enzyme has a low $K_m$ for $NH_4^+$ , making it very efficient even at low ammonia concentrations. - Note: Glutamine is a major source of nitrogen ( $NH_4^+$ ) for myriad other biosynthetic reactions.
Glutamate Synthase Pathway: Under conditions of nitrogen starvation in bacteria and plants, a two-enzyme reaction involving Glutamine synthetase and Glutamate synthase is used to transfer the gamma-amino group to $\alpha \text{-ketoglutarate}$ , yielding two molecules of glutamate.
Serine/Glycine Synthesis: - Precursor: $3 \text{-phosphoglycerate}$ (3PG) from glycolysis. - Conversion: Serine is converted to glycine via Serine hydroxymethyltransferase. This reaction is vital for one-carbon metabolism as it generates $N^5, N^{10} \text{-methylene-THF}$ .
Proline and Ornithine: - Synthesized from $\alpha \text{-ketoglutarate}$ via glutamate and glutamic semialdehyde. - This process is essentially the reverse of their catabolism (which involves oxidation and loss of nitrogen), whereas synthesis involves reduction and gain of nitrogen.

Essential Amino Acids and Chirality

Essentiality Principle: Amino acids that humans cannot synthesize (essential) tend to have complex, difficult biosynthetic pathways. Non-essential amino acids tend to be synthesized via "easy," short pathways.
Arginine ( $R$ ): While categorized as non-essential in adults, it must be considered essential for infants and children who require more arginine for growth than the urea cycle can provide.
Basis of L-Chirality: - During transamination of an $\alpha \text{-keto acid}$ , the spatial arrangement of the cofactors and residues in the enzyme active site—specifically Pyidoxal Phosphate (PLP) on the left, Arginine (Arg) on the right, and Lysine (Lys) underneath—ensures that the proton is transferred such that the resulting amino acid is always in the L-configuration.

One-Carbon Metabolism: Tetrahydrofolate (THF)

THF Structure and Origin: - Tetrahydrofolate is a reduced form of folic acid (Vitamin $B_9$ ). - It consists of three components: a pteridine ring, para-aminobenzoate (PABA), and glutamate. - Dihydropteroate synthase: Found in bacteria; synthesizes folate.
Inhibition and Medicine: - Sulfa Drugs (Sulfanilamide): Structural analogs of PABA that competitively inhibit dihydropteroate synthase. They are safe for humans because we do not synthesize folate, but must acquire it from the diet. - Resistance: Bacteria can become resistant to sulfa drugs by evolving the ability to take up folate from their environment.
Folate Derivatives: THF can carry one-carbon units at different oxidation states at positions $N^5$ , $N^{10}$ , or bridged between both. - Nomenclature Checklist: $N^5 \text{-methyl-THF}$ , $N^5, N^{10} \text{-methylene-THF}$ , $N^5, N^{10} \text{-methenyl-THF}$ , $N^{10} \text{-formyl-THF}$ , and $N^5 \text{-formiminothiol-THF}$ . - Methyl groups ($-CH_3$): Typically carried by SAM. - Carboxy groups ( $CO_2$ ): Carried by Biotin.
Serine Hydroxymethyltransferase: This is the single largest source of one-carbon units for THF, converting Serine to Glycine while transferring a carbon to THF to form $N^5, N^{10} \text{-methylene-THF}$ .

Activated Methyl Cycle and SAM

S-adenosylmethionine (SAM): The primary methyl donor in the cell. - Synthesis: SAM is formed from Methionine and ATP by SAM synthetase. - Energy Cost: Very high; $\text{ATP} \rightarrow \text{Adenosine} + 3 P_i$ . The sulfur of methionine attacks the $C_{5'}$ of ATP. - Transfer Potential: The trivalent positively charged sulfur makes the methyl group highly reactive, giving it a higher transfer potential than $N^5 \text{-methyl-THF}$ .
The Cycle: 1. SAM donates its methyl group to an acceptor (DNA, protein, small molecules like norepinephrine). 2. The remaining molecule is S-adenosylhomocysteine. 3. This is hydrolyzed to adenosine and homocysteine. 4. Methionine Synthase (Homocysteine Methyltransferase): Homocysteine is methylated back to methionine using $N^5 \text{-methyl-THF}$ and Vitamin $B_{12}$ .
Vitamin $B_{12}$ Dependance: Only two reactions in mammals require $B_{12}$ : Methionine Synthase and methylmalonyl CoA mutase (the two enzymes are homologous).
Formyl-Methionine: In bacteria, the first amino acid in protein synthesis is $N \text{-formyl-methionine}$ , requiring formyl-THF.

Specialized Amino Acid Derivatives

Glutathione (GSH): A tri-peptide crucial for maintaining a reducing environment. - Redox Action: $2 \text{GSH} + \text{ROOH} \rightarrow \text{GSSG} + \text{ROH} + H_2O$ via Glutathione peroxidase. - Regeneration: Glutathione reductase uses $NADPH + H^+$ to reduce the disulfide bond in GSSG back to two GSH.
Selenocysteine: - Glutathione peroxidase contains selenocysteine, where selenium replaces sulfur. - Selenium is essential in trace amounts but toxic in higher quantities. - Agricultural Case Study: In New Zealand, selenium-deficient soil caused cardiovascular disease in sheep until hay was sprayed with selenium salts.
Heme Biosynthesis: - Precursors: Succinyl CoA and Glycine. - Intermediate: $\delta \text{-aminolevulinate}$ , which leads to a tetrapyrrole (precursor to heme, chlorophyll, and cyanocobalamin).
Heme Breakdown: - Heme is degraded into biliverdin (green) and then reduced to bilirubin (yellow). - Bilirubin is a linear tetrapyrrole found in bruises and is the cause of jaundice (yellow sclera or skin).

Nucleotide Metabolism: Purines

Nomenclature: - Base: Hypoxanthine; Nucleoside: Inosine; Nucleotide: Inosinate (IMP). - Base: Adenine; Nucleoside: Adenosine; Nucleotide: Adenylate (AMP). - Base: Guanine; Nucleoside: Guanosine; Nucleotide: Guanylate (GMP).
De Novo Synthesis: - Occurs in the cytosol. - Synthesized on a platform of PRPP (Phosphoribosyl Pyrophosphate). - PRPP Synthetase: Converts Ribose $5 \text{-phosphate} + ATP \rightarrow PRPP + AMP$ . - IMP Pathway: 9 steps are required to convert PRPP to Inosinate (IMP). IMP is the parent purine. - AMP and GMP: Synthesis from IMP occurs in two steps. One requires $ATP$ ; the other requires $GTP$ .
Deoxyribonucleotide Synthesis: - Ribonucleotide Reductase (RNR): Converts ribo-NDPs to deoxy-NDPs (at the diphosphate level). - Requires thioredoxin and NADPH. - Kinases: Mononucleotides have specific kinases (e.g., AMPK, GMPK). There is only one Nucleoside Diphosphate Kinase (NDK) with broad specificity for all Ribo- and Deoxy-NDPs/NTPs.

Nucleotide Metabolism: Pyrimidines

De Novo Synthesis Characteristics: 1. The pyrimidine ring is synthesized independently before the attachment of ribose. 2. Carbamoyl Phosphate Synthetase II (CPS II): Cytoplasmic enzyme that uses Glutamine as the ammonia donor. 3. CAD Enzyme: A single large protein with three activities: Carbamoyl phosphate synthetase, Aspartate transcarbamoylase, and Dihydroorotase. 4. Orotate: The first major intermediate. 5. Orotidylate (OMP): Formed by combining orotate and PRPP. Decarboxylation of OMP yields Uridylate (UMP).
CTP Formation: CTP is synthesized by amination of UTP using Glutamine and ATP via Cytidine triphosphate synthetase.

Thymidylate Cycle and Clinical Inhibition

Thymidylate Synthase: Converts $dUMP \rightarrow dTMP$ . - It uses $N^5, N^{10} \text{-methylene-THF}$ as the methyl donor. - Unique Reaction: Methylene-THF gives up a hydride, resulting in its oxidation to Dihydrofolate (DHF).
Dihydrofolate Reductase (DHFR): Regenerates THF from DHF using $NADPH$ .
Cancer Treatment Targets: - Methotrexate (and Aminopterin): Structural analogs of DHF that competitively inhibit DHFR, stopping DNA synthesis by depleting THF pools. - DHFR Gene Amplification: Cancer cells can become resistant to methotrexate by increasing the number of DHFR gene copies. - 5-Fluorodeoxyuridylate (5-FU): A suicide inhibitor of Thymidylate Synthase. It forms a permanent tertiary adduct with the enzyme and the folate cofactor.
Antibiotics: Trimethoprim binds $10^5 \text{-fold}$ more tightly to bacterial DHFR than to human DHFR.

Salvage Pathways and Degradation

Purine Salvage: - More energy-efficient than de novo synthesis. - HGPRT: Recombines PRPP with Guanine (to GMP) or Hypoxanthine (to IMP). - APRT: Recombines PRPP with Adenine (to AMP). - Lesch-Nyhan Syndrome: Caused by a mutation in HGPRT, resulting in severe mental retardation.
Pyrimidine Salvage and Antivirals: - Thymidine Kinase (TK): Converts Thymidine to TMP. - Acyclovir: Guanosine analog used for Herpes (HSV). Viral TK binds it 200 times better than human TK. Once phosphorylated, it incorporates into viral DNA and acts as a chain terminator because it lacks a $3' \text{-OH}$ . Similar strategy for AZT in HIV treatment.
Purine Degradation and Gout: - Sequence: $AMP \rightarrow \text{Inosine} \rightarrow \text{Hypoxanthine} \rightarrow \text{Xanthine} \rightarrow \text{Uric Acid} \rightarrow \text{Urate}$ . - Xanthine Oxidase (XO): Catalyzes the steps from hypoxanthine to urate. It requires molybdenum. - Gout: High plasma levels of urate lead to crystallization in joints. - Allopurinol: An analog of hypoxanthine and another suicide inhibitor. It is oxidized by XO to oxipurinol, which binds irreversibly to the active site, decreasing urate levels.