1. Chapter 17 (Part 1) Amino Acid Metabolism: Nitrogen Assimilation and Amino Acid Biosynthesis

2. Nitrogen Assimilation Nitrogen is required in the synthesis of amino acids, purine and pyrimidine nucleotides, and a number of other important biological compounds. Organisms need to obtain nitrogen in a usable form. Nitrogen in the form of ammonia is assimilated by biological systems Nitrogen is originally assimilated from the environment by microorganisms and plants. Animals must obtain biological forms of nitrogen from their diets.

3. Nitrogen Fixation Most abundant form of nitrogen on Earth is N 2 gas (makes up 80% of air) N 2 gas is very stable and inert. 2 N connected by triple bond (225 kcal/mole required to break bond). N 2 gas can be converted to biologically accessible forms in three ways: 1)N 2 can be reduced to NO 3 - by lightning and UV radiation (15% of fixed nitrogen) 2)N 2 can be reduced to NH 3 through industrial processes (25% of total fixed nitrogen) Requires temperatures of 500 o C and 300 atm) 3)N 2 can be reduced to NH 3 by nitrogen fixing bacteria (60% 0f total fixed nitrogen)

4. Nitrogen Cycle

5. Biological Nitrogen Fixation Process performed only by special free living (cyanobacteria) microorganims. Nitrogen fixation can also be performed by microorganisms (Rhizobium, Bradyrhizobium) that exist as symbiotes with specific plant species (Legumes – soybean, alfalfa) N 2 is converted to NH 3 in a reaction catalyzed by the nitrogenase complex.

6. Nitrogenase consist of two subunits, an Fe-protein (reductase) and an MoFe-protein (nitrogenase). Reductive process: N 2 + 8e - + 8H +  2 NH 3 + H 2 For every e - used in the process 2 ATP are consumed, so 16 ATPs are required to convert 1 N 2 to NH 3 Enzymes are highly sensitive to O 2. Require anerobic conditions

7. Nitrate and Nitrite to Ammonia NO 3 - and NO 2 - must be converted to NH 3 to be assimilated into organisms. Process referred to as nitrification Requires two enzymes nitrate reductase and nitrite reductase

8. Ammonia Assimilation Glutamate dehydrogenase - reductive amination of  -ketogluturate to glutamate. Glutamine synthetase - ATPdependent amidation of gamma-carboxyl of glutamate to glutamine

9. Glutamate Dehydrogenase Impt. in bacteria when ammonia is present at high concentrations. In animal and plant systems enzyme is localized within the mitochondria. The enzyme is near equilibrium and runs in the direction toward glutamate catabolism (anaplerotic rxn to regenerate  -ketogluturate).

10. Glutamine Synthetase (GS) Glutamine plays important role in N- metabolism. Acts a NH 3 donor for purine and pyrimidine synthesis In mammals, glutamine is synthesized in muscle tissue using GS and transported to other tissues.

11. Glutamine back to Glutamate Under low NH 3 conditions, in conjunction with glutamine synthetase, bacteria use glutamate synthase to convert Gln and  -ketogluturate to 2 glutamates. The Km of glutamate dehydrogenase for NH3 is high, the Km of glutamine synthetase is low.

12. Glutamine Synthetase GS in bacteria is regulated in three ways: –Feedback inhibition –Covalent modification (interconverts between inactive and active forms) –Regulation of gene expression and protein synthesis control the amount of GS in cells

13. Allosteric Modulation of GS Acitivity in Bacteria

14. Allosteric Modulators of Mammalian GS Activator –  -ketogluturate Inhibitors – glycine, serine, alanine, carbamyl-phosphate

15. Regulation of Bacterial GS by Covalent Modification

16. Gene GlnA is actively transcribed only if transcriptional enhancer NR I is in its phosphorylated form, NR I -P NR I is phosphorylated by NR II, a protein kinase If NR II is complexed with the activated form of P II acts as a phosphatase, not a kinase

17. Glutamate serves as primary N- donor for AA synthesis through transamination reactions

18. Transamination rxns involve Pyridoxol-phosphate and formation of Schiff Base

19. Essential vs. Non-Essential AA’s Plants and bacteria can synthesize all 20 amino acids. Animals must obtain 8 amino acids (essential AA’s) from diet

20. Amino Acid Biosynthesis is a Diverse Process