Chapter 22 Biosynthesis of amino acids, nucleotide

1. For Biochemistry II, Dec. 16 and 31, 2009 Chapter 22Biosynthesis of amino acids, nucleotide To be lectured by Professor Zengyi Chang 1. Source of nitrogen. 2. Source of carbon. 3. De novo and salvage pathways. 4. Ways to balance the synthesis of each.

2. Overview

3. The 20 standard amino acids are usually categorized into five families Issues: What are used as precursors to generate the carbon skeletons what are the chemical processes and enzymes involved How are the processes related to each other Why only L-amine acids are synthesized in the cells How would a balanced synthesis of each amino acid be achieved

4. Tyrosine cysteine

5. The eight nucleotides found in DNA and RNA Issues: How are the base, the sugar, & the phosphate assembled What are the starting precursors What chemical processes and enzymes are involved Are the synthetic processes related to each other How would a balanced synthesis of each to be achieved

6. Nutritional requirements for amino acids in mammals The biosyntheses of the 20 amino acids can be grouped into six families ( the a-keto acid not synthesized!) Amino acids also function as precursors to hormones, coenzymes, porphyrins, pigments, Neurotransmitters, etc. Nutritional quality of proteins for humans: Mammals > fish & poultry> fruits & plants.

7. The biosynthesis of nucleotides: an outline Gln + HCO3- The pyrimidine ring is assembled first before attached to Ribose phosphate. The purine ring is assembled on ribose phosphate.

8. Difficulties and importance of studying this chapter • Many pathways involve many steps and intermediates. • Some most unusual chemical transformations in biosystems found here. • Many genetic diseases are caused by defects of enzymes discussed here. • Many pharmaceuticals in common use to combat infectious diseases or cancer are inhibitors of enzymes discussed here. • Best-understood examples of enzyme regulation are seen here.

9. Most organisms maintain strict economy in their use of ammonia, amino acids and nucleotides • Biologically useful nitrogen compounds are generally scarce in the natural environments. • Free amino acids, pyrimidines and purines formed from metabolic turnover are often salvaged (reused). • Only certain bacteria are able to fix N2 into ammonia (NH3 or NH4+).

10. Biosynthesis of Amino acids and nucleotides are closely related • Nitrogen arises from common biological sources (N2 fixation). • The two sets of pathways are extensively intertwined (shared intermediates). • Much common chemistry are found in both pathways: transfer of nitrogen (often from Gln) or one-carbon units (carried on tetrahydrofolate).

11. From where does nitrogen come from

13. Relationships between Inorganic and organic nitrogen metabolism Few organisms can use the N2 in air, and many soils are poor in nitrate: Nitrogen bioavailability limits growth for most organisms (thus the world’s food supply)!

14. Nitrogen (azote) enters biomolecules via amino acids (revealed by using radio isotopes, 15N and 14C) Certain bacteria N2 → ammonia →Gln/Glu →other biomolecules Highly comparable with CO2 fixation: CO2 → 3-phospohglycerate → hexose → other biomolecules. Both are highly energy consuming, needing NADPH and ATP!

15. N2 fixation is thermodynamically favorable, kinetically extremely slow Has a bond energy of 930 kJ/mol (while that for a C-O is 350 kJ/mol) Biological N2 fixation in diazotrophs: N2+8H++8e−+16ATP → 2NH3+H2+16ADP+16Pi Here ATP hydrolysis reduces the heights of the activation energy barrier, instead of for thermodynamical purposes. The precise number of ATP consumed in this process has not yet been established.

16. Biological nitrogen fixation was first discovered by Martinus Beijerinck, a Dutch microbiologist (1886). Nitrogen fixation is catalyzed by the nitrogenase complex, present only in certain bacteria (diazotrophs like cyanobacteria and rhizobia) and energetically costly. Cyanobacteria Rhizobia The Haber method: N2 +3H2 2NH3 G`o = - 33.5kJ/mol with iron catalyst, 500oC, 300 atm. Can we design a process of producing ammonia under milder condition by learning from what bacteria do in fixing nitrogen?

17. Nitrogenase reductase (Fe Protein) Nitrogenase (MoFe Protein) The nitrogenase complex ATP binding and hydrolysis is thought to both drive the reduction of the P-cluster & to trigger a conformational change in the reductase that causes it to dissociate transiently from the nitrogenase, assuring unidirectional electron flow. Electron Donors (ferredoxn or Flavodoxin) 8e− are needed to reduce each N2.

18. The nitrogenase complex is extremely labile to O2 and various protective mechanisms have evolved: living anaerobically, forming thick walls, uncoupling e- transport from ATP synthesis (entering O2 is used immediately) or being protected by O2-binding proteins (e.g., leghemoglobin).. Cyanobacteria Rhizobia heterocyst

19. Reduced nitrogen in the form of NH4+ is assimilated into amino acids mainly via a two-enzyme pathway : glutamine synthetase and glutamate synthase(an enzyme only present in bacteriaand plants).

20. Ammonia enters organic compounds in bacteria and plants mainly via Gln and Glu Gln synthetase (present in all organisms) Act to detoxify ammonia in animals! Glu synthase (present only in bacteria and plants) The combined action of Gln synthetase and Glu synthase leads to the net synthesis of Glu from a-ketoglutarate and NH4+!

21. The Glutamine synthetase is a primary regulatory point in nitrogen metabolism: being regulated by at least eight allosteric effectors and reversible adenylylation in prokaryotes. The glutamine synthesis is constantly tailored to cellular needs!

22. The E. coliglutamine synthetase has 12 subunits (dodecamers) arranged as two rings of hexamers. Active sites at interfaces Mn Tyr397 (adenylylation site)

23. The glutamine synthetaseis cumulatively inhibited by at least 8 allosteric effectors, mostly end products of glutamine metabolism. Each of the 50 kDa subunit contains binding sites for all the 8 allosteric effectors in addition to the active sites!

24. AMP Gln synthetase The inactive form Adenylylation increases the sensitivity of each subunit to the 8 allosteric inhibitors. Tyr397 A specific Tyr residue in bacterial glutamine synthetase can be reversibly adenylylated by the catalysis of adenylyltransferase (AT), whose activity is modulated by a regulatory protein (PII), whose activity is in turn regulated by uridylylation, catalyzed again by a single enzyme (uridylyltransferase, UT).

25. The activity of E. coli Gln synthetase is regulated by reversible adenylylation. Adenylyl- transferase Uridylyl- transferase Adenylyl- transferase “Activated nitrogen” Consequence of the regulation: high Gln level → low Gln synthetase activity; High a-ketoglutarate→ high Gln synthetase activity. The animal Gln synthetase seems to be regulated by changing its oligomeric status (octameric to tetrameric).

26. Amidotransferases: a family of enzymes that catalyze the donation of the amide amino group from Gln to many other “acceptor” compounds.

27. The biosythesis of 20 standard amino acids

28. Highly conserved Two-domain enzymes Varies A proposed general action mechanism for amidotransferases.

29. The carbon skeletons of the 20 amino acids (in L-configuration) are derived mainly from intermediates of glycolysis, citric acid cycle, and pentose phosphate pathway in bacteria and plants.

30. Pathways for synthesizing the “essential” amino acids are usually complex, involving 5-16 steps.

31. Pyridoxal phosophate and tetrahydrofolate are two cofactors widely used in amino acid metabolism Tetrahydrofolate (carries one-carbon units) Pyridoxal phosophate