Anabolism III - Biosynthesis Amino Acids and Nucleotides

Biosynthesis Amino Acids & Nucleotides H2O–M H+ H+ e– H2O–M–H H+ e – M H+ + HO– + M–H2 e– e– H+ Diimino H+ e– H+ e– H2O + H2 Hydrazine H+ e– H+ Redox Reactions in Dinitrogenase e– • The net reaction of the nitrogenase complex: N2 + 8 H+ + 8 e– + 16 ATP = 2 NH3 + H2 + 16 ADP + 16 Pi • Mechanism of dinitrogenase is poorly understood. –Plausible mechanism involving the Fe-Mo cofactor (M) binding directly to N2. –Extremely complex redox reaction that involves several metal atoms as cofactors and/or electron transporters 1 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 5) Control of nitrogen metabolism 6) Biosynthesis and degradation of heme; other 2° products of amino acids Biosynthesis Amino Acids & Nucleotides Two Important Enzymes in Nitrogen Assimilation by PLANTS 1. Nitrate reductase NO3– + 2 e– à NO2– • • • large, soluble protein contains novel Mo cofactor e– from NADH 2. Nitrite reductase NO2– + 6 e– à NH4+ • • found in chloroplasts in plants: e– comes from ferredoxin (Fd) in non-photosynthetic microbes: e– comes from NADPH Amino acids 2 Biosynthesis Amino Acids & Nucleotides Problem: need to get new ammonia onto the a-carbon for transamination Ammonia is Incorporated into Biomolecules in PLANTS & ANIMALS Through Glu and Gln in 2 steps. •Glutamine is made from Glu by glutamine synthetase in a SUM: two-step process (we discussed this previously when moving ammonia from extrahepatic tissues). Glutamate + ATP → γ-glutamyl-phosphate + ADP γ-glutamyl-phosphate + +NH4 → glutamine + Pi + H+ Glutamate + +NH4 + ATP → glutamine + ADP + Pi + H+ •Glutamate is made from Gln α-Ketoglutarate + glutamine + NADPH + H+ → 2 glutamate + NADP+ and α-Ketoglutarate by glutamate synthase (GOGAT).α-Ketoglutarate + +NH4 + ATP + NADPH + H+ → glutamate + NADP+ + ADP + Pi α-Ketoglutarate, an intermediate of the citric acid Assimilation! cycle, undergoes reductive But, let’s discuss GlySynthease…. amination with glutamine as (An alternative name for this enzyme, glutamate:oxoglutarate aminotransferase, yields the acronym GOGAT, by which the enzyme also is known.) nitrogen donor. Biosynthesis Amino Acids & Nucleotides What about GOGAT…. Structure of Gln Synthetase (a12) 50 kDa 3 Biosynthesis Amino Acids & Nucleotides GOGAT, and many other Gln Amidotransferases have similar mechanisms: • There are two domains: • one binds Gln • other is amino group acceptor and binds substrate Cys acts as nucleophile to cleave amide bond of Gln àForms glutamyl-enzyme intermediate àNH4 goes thru channel • Then second substrate binds to accept NH4 • In the case of GOGAT, there is another subunit to provide for the reduction R 1–CO O – R 2–CH 2CH 2CO O – a-ketoglutarate = O R 2–C–CO O – à = NH2 R 2–C–CO O – à Glutamate Biosynthesis Amino Acids & Nucleotides Mechanism of Glu Synthase: GOGAT a-Subunit (site 3) Gln Tunnel Glu #1 a-Subunit (site 2) (a-KG) a-Subunit (160 kDa) b-Subunit (50 kDa) b-Subunit (site 1) 3Fe4S Glu #2 α-Ketoglutarate + glutamine + NADPH + H+ → 2 glutamate + NADP+ 4 Biosynthesis Amino Acids & Nucleotides Ammonia assimilation • Glutamine Synthetase • Glutamate Synthase: GOGAT Net: a-ketoglutarate + +NH4 à Glutamate (ATP and NADPH needed) 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 5) Control of nitrogen metabolism 6) Biosynthesis and degradation of heme; other 2° products of amino acids 5 Biosynthesis Amino Acids & Nucleotides Amino Acid Synthesis Overview 6 •Source of N is Glu or NH4+ (via Gln) •Derive from intermediates of: – glycolysis – citric acid cycle – pentose phosphate pathway •Bacteria can synthesize all 20. •Mammals require some in diet: –Essential –Non-essential 6 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 Biosynthesis Amino Acids & Nucleotides Transaminase route Amidation route AMP + PPi GPTA Gln + ATP Asparagine Synthetase GOTA + + GOTA Glutam ine Synthetase ADP NH4+ +ATP 7 Biosynthesis Amino Acids & Nucleotides Glu Family: Pro (Arg) NADP+ + H+ D1 -pyrroline-5-carboxylate reductase NADPH In mammals, Glu is only reduced in intestines. Then the semialdehyde goes to Orn and through Urea Cycle to Arg. In most tissues, it is converted to Arg using the degradation pathway for Pro. oxaloacetate GOTA aspartate In bacteria, Glu is acetylated. This limits the un-catalyzed cyclization of the Glu-semialdehyde before its converted to Orn. Biosynthesis Amino Acids & Nucleotides 3-PGA Family: Ser Serine Derives from 3-Phosphoglycerate of Glycolysis NAD+ phosphoglycerate dehydrogenase Pi phosphoserine phosphatase NADH H2O phosphoserine aminotransferase PLP • Oxidation à transamination à to yield serine dephosphorylation 8 Biosynthesis Amino Acids & Nucleotides 3-PGA Family + From Essential Family: Gly, Cys Serine contributes to Glycine and Cysteine Glycine is reverse of degradation Cysteine carbons are from Ser Bacterial/plants Sulfation through APS In bacteria and plants, sulfates are the source of S through APS. In mammals, the Cys-sulfur is recycled from methionine degradation…... Biosynthesis Amino Acids & Nucleotides 3-PGA Family + From Essential Family: Cys, Tyr Biosynthesis of Cys from Homocysteine and Ser in Mammals The sulfur for Cysteine and nearly all of Tyrosine come from Essential Amino Acids via same enzymes used in degradation 9 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 Biosynthesis Amino Acids & Nucleotides Asp/Pyruvate Family: Lys, Met, Thr, Ile, Val, Leu Oxaloacetate Yields Asp, which Yields Met and Thr, which along with Pyruvate Yields Lys, Val, Ile and Leu, which needs Acetyl-CoA Acetyl-CoA 10 NH 4+ | –OOC-C-C-COO– Asp/Pyruvate Family Asp 4Asp + 5Pyr + 1Acetyl-CoA + 6ATP + 6NADPH + 2NAD+ + 2 H+ + Me-THF à Lys + Met + Thr + Ile + Val + Leu + 5NADP+ + 5H+ + 6Pi + 4CO2 + 2NADH + 4H2O + THF ATP O || NH 4+ | NADPH P O || Asp-b-semialdehyde Specific for anabolism P –O-C-C-C-COO– HO-C-C-C-COO– SB/dehy O || –OOC-C-C Homoserine H2O NADPH Pyr Succ-CoA P C-C-C-COO– | NADPH NH 4+ | 2H2O 4+ O OH NH | || | –OOC-C-C-C-C-C-COO– NH4+ HC-C-C-COO– ATP NH 4+ | HO NH4+ | | Asp-b-phosphate P –C-C-C-COO– Same as in catabolism Pyr Thr NH4+ CO2 C-C-C-COO– a-ketobutyrate Pyr NH 4+ NH 4+ | | –OOC-C-C-S-C-C-C-COO– || C-C| | | Succ–NH NH4+ | –OOC-C-C-C-C-C-COO– Succ | NH4+ N5-m ethyl-THF NH 4+ NH 4+ | | –OOC-C-C-C-C-C-COO– NH 4+ | H3C-S-C-C-C-COO– | | C-C–OH | C C-C-C–OH | C O=C-COO– | C-C-C | C Glu C-COO– | C-C-C | C Ile | C-COO– Val AcCoA O=C-COO– NH 4+ | | C-C | C Glu Lys CO2 NH4+ | –OOC-C-C-C-C-C-NH4+ O || C-C-CoA H2O NH 4+ | PLP Met HO–C-COO– HO–C-COO– Succ–NH NH4+ | –OOC-C-C-C-C-C-COO– | HS-C-C-C-COO– hom ocysteine C=O | C NADPH B12 Glu Pyr + NH4+ NH 4+ | C-C-COO– TPP | C=O | C Cystathionine C-C-COO– HO | O HO || –OOC-C-C-C-C-C-COO– | Cys Succ O || C-C-C-COO– O Succ-O-C-C-C-COO– || C-C-COO– O || Succ-CoA NH 4+ | O – C-COO | HO–C-COO– | C-C C-C | | C C a -ketoisovalerate – HO–C-COO | C-COO– | C-C | C NAD+ O=C-COO– CO2 | C | C-C | C Glu NH4+ | C-C-C-C-COO– | C Leu 11