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

Chapter 17 (Part 1)

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 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:

1) N2 can be reduced to NO3- by lightning and UV radiation (15% of fixed nitrogen)

2) N2 can be reduced to NH3 through industrial processes (25% of total fixed nitrogen) Requires temperatures of 500oC and 300 atm)

3) N2 can be reduced to NH3 by nitrogen fixing bacteria (60% 0f 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)

• N2 is converted to NH3 in a reaction catalyzed by the nitrogenase complex.

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

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

Ammonia Assimilation• Glutamate dehydrogenase -

reductive amination of -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 -ketogluturate).

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.

Glutamine back to Glutamate•Under low NH3 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.

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 – -ketogluturate• Inhibitors – glycine, serine,

alanine, carbamyl-phosphate

Regulation of Bacterial

GS by Covalent

Modification

• 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

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