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

2. Overview

3. 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 The 20 standard amino acids are usually categorized into five families

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

7. The biosynthesis of nucleotides: an outline Gln + HCO 3 - The purine ring is assembled on ribose phosphate. The pyrimidine ring is assembled first before attached to 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 N 2 into ammonia (NH 3 or NH 4 + ).

10. Biosynthesis of Amino acids and nucleotides are closely related Nitrogen arises from common biological sources (N 2 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 N 2 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, 15 N and 14 C) N 2 → ammonia → Gln/Glu →other biomolecules Certain bacteria Highly comparable with CO 2 fixation: CO 2 → 3-phospohglycerate → hexose → other biomolecules. Both are highly energy consuming, needing NADPH and ATP!

15. N 2 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 N 2 fixation in diazotrophs: N 2 +8H + +8e − +16ATP → 2NH 3 +H 2 +16ADP+16P i 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. Nitrogen fixation is catalyzed by the nitrogenase complex, present only in certain bacteria (diazotrophs like cyanobacteria and rhizobia) and energetically costly. The Haber method: N 2 +3H 2 2NH 3  G` o = - 33.5kJ/mol with iron catalyst, 500 o C, 300 atm. Cyanobacteria Rhizobia Biological nitrogen fixation was first discovered by Martinus Beijerinck, a Dutch microbiologist (1886). Can we design a process of producing ammonia under milder condition by learning from what bacteria do in fixing nitrogen?

17. The nitrogenase complex Nitrogenase (MoFe Protein)Nitrogenase reductase (Fe Protein) Electron Donors (ferredoxn or Flavodoxin) 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. 8e− are needed to reduce each N 2.

18. The nitrogenase complex is extremely labile to O 2 and various protective mechanisms have evolved: living anaerobically, forming thick walls, uncoupling e - transport from ATP synthesis (entering O 2 is used immediately) or being protected by O 2 - binding proteins (e.g., leghemoglobin).. heterocyst Cyanobacteria Rhizobia

19. Reduced nitrogen in the form of NH 4 + is assimilated into amino acids mainly via a two-enzyme pathway : glutamine synthetase and glutamate synthase (an enzyme only present in bacteria and plants).

20. Ammonia enters organic compounds in bacteria and plants mainly via Gln and Glu Gln synthetase Glu synthase(present only in bacteria and plants) (present in all organisms) Act to detoxify ammonia in animals! The combined action of Gln synthetase and Glu synthase leads to the net synthesis of Glu from  -ketoglutarate and NH 4 + !

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. coli glutamine synthetase has 12 subunits (dodecamers) arranged as two rings of hexamers. Active sites at interfaces Tyr 397 (adenylylation site) Mn

23. The glutamine synthetase is 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. 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 (P II ), whose activity is in turn regulated by uridylylation, catalyzed again by a single enzyme (uridylyltransferase, UT). Gln synthetase The inactive form Adenylylation increases the sensitivity of each subunit to the 8 allosteric inhibitors. AMP Tyr 397

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

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 Pyridoxal phosophate Tetrahydrofolate (carries one-carbon units)