Amino Acid Biosynthesis and Nucleotides Lecture Note

Biosynthesis Amino Acids & Nucleotides Essential Amino acids: These require many steps and unique to those used for degradation. Asp/Pyruvate Family Aromatic Family Histidine Met Thr Lys Ile Val Leu Phe Trp His 7 5 9 10 4 7 10 12 10 –Asp/Cys/THF/Glu –Asp/Glu –Asp/Pyr/Glu –Asp(Thr)/Pyr/Glu –Pyr/Glu –Pyr/AcCoA/Glu –E4P/PEP/Glu –E4P/PEP/Gln/R5P/Ser –R5P/ATP/Gln/Glu 1 Biosynthesis Amino Acids & Nucleotides Aromatic Family: Phe, Trp Aromatic Amino Acids Derive from PEP and Erythrose 4Phosphate (and Rib5P and Ser) • Very complicated and amazing chemistry! • Rings must be synthesized and closed and then oxidized to create double bonds. • Chorismate is a common intermediate. Ribose 5-phosphate Serine Biosynthesis of Chorismate, a Key Intermediate in Aromatic Amino Acid Biosynthesis Chroism ate 2 Biosynthesis Amino Acids & Nucleotides Aromatic Family: Phe, Trp prephenate dehydratase TA CO2 + H2O chorismate mutase NADPH + PEP CO2 NADP+ prephenate dehydrogenase TA anthranilate synthase E4P Tryptophan Biosynthesis Amino Acids & Nucleotides Trp Synthase PEP E4P Tryptophan 3 Histidine Derives from Ribose 5-Phosphate Ribose 5-Phosphate ATP Ribose Phosphate Pyrophosphokinase AMP PRPP •Synthesized from ribose 5phosphate via ribose phosphate pyrophosphokinase – a highly regulated allosteric enzyme Biosynthesis Amino Acids & Nucleotides Essential vs. Nonessential and Conditionally Essential Amino Acids • Essential amino acids must be obtained as dietary protein. • Nonessential amino acids are easily made from central metabolites. TABLE 18-1 Nonessential and Essential Amino Acids for Humans and the Albino Rat Nonessential Alanine Asparagine Aspartate Conditionally essentiala Arginine Cysteine Glutamine Essential Histidine Isoleucine Leucine Glutamate Serine Glycine Proline Tyrosine Lysine Methionine Phenylalanine Threonine Tryptophan Valine aRequired to some degree in young, growing animals and/or sometimes during illness. • Consumption of a • Conditionally Essential amino acids variety of foods are made from essential, or become supplies all the essential in certain physiological essential amino acids. conditions. 4 Biosynthesis Amino Acids & Nucleotides Non-essential Amino acids: These are very few steps and often the same Arg-Val-His-Ile-Leu-Lys-Met-Phe Thr-Trp enzyme(s)same used for degradation. Professor A.V.HILL M.P. was a Tea Totaller as AA #steps degradation From? Asp Glu Ala Asn Gln Pro Ser Gly Cys Tyr 1 1 1 1 1 3(1) 3 1 2 1 ✓ ✓ ✓ – – (✓) – ✓ ✓ ✓ Red=biosynthesis specific OAA a-KG Pyr Asp Glu Glu/Arg 3PGA Ser Ser/Met Phe Transaminase route Amidation route Glu Family 3-PGA Family From Essential Family Green=essential ANABOLISM III: Biosynthesis Amino Acids & Nucleotides 1) Nitrogen fixation: N2 à NH4 2) Nitrogen assimilation: incorporation of ammonia into biomolecules 3) Biosynthesis of amino acids a) non-essential b) essential 4) Biosynthesis of nucleotides a) sources b) de novo purines (R)(as nucleotides*); salvage; regulation c) de novo pyrimidines (Y)(as bases); making nucleotide; regulation d) deoxy-ribonucleotides, dTMP, and phosphorylation to NTP & dNTP e) regulating levels for DNA synthesis 5) Control of nitrogen metabolism 6) Biosynthesis and degradation of heme; other 2° products of amino acids *Bases synthesized while attached to ribose-5-P; products are RMP (R is one-letter code for purine, Y is one letter code for pyrimidine) 5 Biosynthesis Amino Acids & Nucleotides Catabolism Anabolism Purines, pyrimidines, Nucleotides porphorins neurotransmitters hormones glutathione CO2 cofactors SAM/THF (C1) a- + NH4 [R,Q] [27 other Ns] a-Ketoglutarate N2 Aspartate a-Keto acids Glutamate Sugars Oxaloacetate Fatty acids Biosynthesis Amino Acids & Nucleotides Two major sources of Nucleotides: 1. They can be synthesized de novo (“from the beginning”) • Purine nucleotides: from Gly, Gln(NH3), Asp(NH3), THF, and CO2, and ribose-5-phosphate (PRPP) • Pyrimidine nucleotides: from Asp, carbamoyl-phosphate, and ribose-5phosphate (PRPP) 2. Nucleotides can be salvaged from RNA, DNA, and cofactor degradation and diet. • Recall purines are degraded to uric acid (no energy) but pyrimidines can be oxidized to acetyl-CoA and Succinyl-CoA • Purine salvage is a significant contribution (80-90%) • Interesting: Many parasites (e.g., malaria) lack de novo biosynthesis and rely exclusively on salvage. Therefore, compounds that inhibit salvage pathways are promising antiparasite drugs. 3. Because ATP/ADP are involved in so many reactions and regulation mechanisms, the [nucleotide] are kept low; so cells must continually synthesize them. • This synthesis may actually limit rates of transcription and replication. 4. Unlike amino-acid biosynthesis, conserved in all organisms studied. 6 Biosynthesis Amino Acids & Nucleotides Purines Overview: 1. Build on ribose (PRPP) 2. Branch to AMP & GMP at IMP 3. Many reactions like those seen before Biosynthesis Amino Acids & Nucleotides R5P + ATP Ribose Phosphate Pyrophosphokinase De Novo Biosynthesis of Purines 0. Begins with PRPP synthesis 1. PRPP reacts with Gln (like His with ATP NH2, or anthranilate in Trp syntheCommittal Step 2. Addition of three carbons from glycine by making amide (like Glu synthase; ammonia channel). 3. Add C1 from THF =R 7 Biosynthesis Amino Acids & Nucleotides De Novo Biosynthesis of Purines se 4. FGAR reacts with Gln (like Glu synthase; ammonia channel). 5. Looks like Schiff base, but its an elimination after phosphorylation. Biosynthesis Amino Acids & Nucleotides De Novo Biosynthesis of Purines 6. Typical carboxylase (6a/b in microorganisms) 7. Add Nitrogen of Asp (recall Urea Cycle). 6 6a biotin 6b 6a 7 6 6b 7 8 Biosynthesis Amino Acids & Nucleotides De Novo Biosynthesis of Purines His biosynthesis 8 8. Removal of formate (can act as anaplerotic reaction to keep ATP synthesis) 9. Add C1 from THF 10. Schiff base formation gets ring closure and IMP 9 Total ATP=10(2 for N10-formylTHF) 10 8 9 10 Biosynthesis Amino Acids & Nucleotides Synthesis of AMP and GMP from IMP 11. Add Nitrogen of Asp (recall Urea Cycle) 12. Removal of formate (can act as anaplerotic reaction to keep ATP synthesis) . 12 11 13 14 13. Add water cross imine and oxidize to keto (recall fatty acid oxidation, except at imine not alkene) 14. Add nitrogen from Gln (recall Glu synthase (ammonia channel), and 3rd time we saw use of Gln for this) Note that ATP is used to synthesize GMP precursor, while GTP is used to synthesize AMP precursor. 9 Recall: Nucleotide Degradation Nucleotides: Nucleosides: Bases: Adenine Biosynthesis AminoDegradation Acids & Nucleotides Nucleotide Salvage Catabolism Pathway of Purines Nucleotides: adenine phosphoribosyl transferase hypoxanthine-guaninephosphoribosyl transferase (ARPT) (HGRPT) Nucleosides: PRPP PRPP Bases: Adenine • Over 90% of purine bases are from salvage pathway. • The brain is especially dependent on salvage pathways. • The lack of HGPRT leads to Lesch-Nyhan syndrome with neurological impairment and finger-and-toe-biting behavior. 10 Biosynthesis AminoDegradation Acids & Nucleotides Nucleotide • The lack of HGPRT leads to Lesch-Nyhan syndrome with neurological impairment and finger-and-toe-biting behavior. Salvage Pathway https://mymedia.bu.edu/channel/BI422/81224851 Dr. Kornberg: Biosynthesis Amino Acids & Nucleotides Regulation of Purine Biosynthesis GDP ⊕ Four Major Sites of Allosteric Regulation 1. PRPP synthetase is inhibited by ADP and GDP. 2. Glutamine-PRPP amidotransferase is inhibited by end-products IMP, AMP, and GMP. 3. Excess GMP inhibits formation of xanthylate from inosinate by IMP dehydrogenase. 4. Excess AMP inhibits formation of adenysuccinate from inosinate by adenylsuccinate synthetase. 11 Biosynthesis Amino Acids & Nucleotides Amide N of glutamine Aspartate Pyrimidines Carbamoyl phosphate (CO2 & +NH4) Overview: 1. Orotic acid 2. Add ribose (PRPP), make UMP 3. CTP made from UTP Biosynthesis Amino Acids & Nucleotides ATP ⊕ De Novo Biosynthesis of Pyrimidines • Unlike purine synthesis, pyrimidine synthesis proceeds by first making the pyrimidine ring (in the form of orotic acid) and then attaching it to ribose 5-phosphate using PRPP. • Aspartate and carbamoyl phosphate provide all the atoms for the heterocycle or pyrimidine. The first pyrimidine is Orotate. (OMP) • This is converted to a nucleotide using PRPP, resulting nucleotide (orotidylate; OMP). • OMP is decarboxylated to form uridylate (UMP). • The other pyrimidine nucleotide used in RNA is made at the triphosphate level; UMP is phosphorylated twice to make UTP. • UTP is converted to CTP by amination using Gln similar to making AMP from XMP. ⊕ • The biosynthesis of CTP is the CLASSIC feedback inhibition by the allosteric negative effector (CTP) on ATCase. Also, activation by GTP 12 Biosynthesis Amino Acids & Nucleotides Regulation of Pyrimidine Biosynthesis via Feedback Aspartate Transcarbamoylase Inhibition (ATCase) (a3)2(b2)3 Recall from 421: ATCase is inhibited by end-product CTP and is accelerated by ATP. ANABOLISM III: Biosynthesis Amino Acids & Nucleotides Involvement of ribonucleotide-derivatives in all of biology 13 Biosynthesis Amino Acids & Nucleotides How are Ribonucleic Acid Precursors converted to Deoxyribonucleic Acid GMPàGDPàGTP Precursors? ….....and how is dTTP made? AMPà ADPàATP So far: à UMPàUDPàUTP CDPßCTP Specific kinases, e.g., UMP kinase, GMP kinase, Adenylate kinase etc. GDPàdGDP ADPàdADP UDPàdUDP CDPàdCDP 2’C-OH bond is directly reduced to 2’-H bond …without activating the carbon for dehydration, etc.! catalyzed by ribonucleotide reductase Non-specific kinase, nucleoside diphosphate kinase Very unique enzyme in all of biochemistry – use of free (works on both oxy- and deoxy-ribose nucleosides) radicals (without cofactors) Mechanism: Two H atoms are donated by NADPH and carried by thioredoxin or glutaredoxin to the active site. –Substrates are the NDPs and the products are dNDP. 14