Amino Acid Metabolism Nitrogen Assimilation and Amino Acid

Amino Acid Metabolism: Nitrogen Assimilation and Amino Acid Biosynthesis

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.

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% 0 f total fixed nitrogen)

Nitrogen Cycle

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.

Nitrogenase • consist of two subunits, an Fe-protein (reductase) and an Mo. Fe-protein (nitrogenase). • Reductive process: N 2 + 8 e- + 8 H+ 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

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

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

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).

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.

Glutamine back to Glutamate • Under low NH 3 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 NH 3 is high, the Km of glutamine synthetase is low.

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

Allosteric Modulation of GS Acitivity in Bacteria

Allosteric Modulators of Mammalian GS • Activator – a-ketogluturate • Inhibitors – glycine, serine, alanine, carbamyl-phosphate

Regulation of Bacterial GS by Covalent Modification

• Gene Gln. A 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

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

Transamination rxns involve Pyridoxol-phosphate and formation of Schiff Base

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

Amino Acid Biosynthesis is a Diverse Process