Chapter 17 (Part 1)

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 N2 gas (makes up 80% of air) • N2 gas is very stable and inert. 2 N connected by triple bond (225 kcal/mole required to break bond). N2 gas can be converted to biologically accessible forms in three ways: • N2 can be reduced to NO3- by lightning and UV radiation (15% of fixed nitrogen) • N2 can be reduced to NH3 through industrial processes (25% of total fixed nitrogen) Requires temperatures of 500oC and 300 atm) • N2 can be reduced to NH3 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) • N2 is converted to NH3 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: N2 + 8e- + 8H+ 2 NH3 + H2 • For every e- used in the process 2 ATP are consumed, so 16 ATPs are required to convert 1 N2 to NH3 • Enzymes are highly sensitive to O2. Require anerobic conditions

7. Nitrate and Nitrite to Ammonia • NO3- and NO2- must be converted to NH3 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 a-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 a-ketogluturate).

10. Glutamine Synthetase (GS) • Glutamine plays important role in N-metabolism. • Acts a NH3 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 NH3 conditions, in conjunction with glutamine synthetase, bacteria use glutamate synthase to convert Gln and a-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 – a-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 NRI is in its phosphorylated form, NRI-P • NRI is phosphorylated by NRII, a protein kinase • If NRII is complexed with the activated form of PII 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. Plants and bacteria can synthesize all 20 amino acids. Animals must obtain 8 amino acids (essential AA’s) from diet Essential vs. Non-Essential AA’s

20. Amino Acid Biosynthesis is a Diverse Process