Nitrogen Acquisition and Amino Acid Metabolism Biochemistry II: Chapter 8 Nitrogen Acquisition and Amino Acid Metabolism Soybean plants can meet their nitrogen requirements both by assimilating nitrate and, in symbiosis with bacteria, fixing N2.

Outline Which metabolic pathways allow organisms to live on inorganic forms of nitrogen? What is the metabolic fate of ammonium? What regulatory mechanisms act on Escherichia coli glutamine synthetase? How do organisms synthesize amino acids? How does amino acid catabolism lead into pathways of energy production?

And Amino Acid Metabolism Dynamics of Protein And Amino Acid Metabolism Dietary Proteins Digestion to Amino Acids Transport in Blood to Cells Protein Synthesis Functional Proteins Protein Degradation In Proteasomes Following Tagging With Ubiquitin Amino Acids Metabolites

How Do the Various Organic Forms of Nitrogen Arise? Metabolism of the 20 common amino acids is considered from the origins and fates of their: (1) Nitrogen atoms (2) Carbon skeletons For mammals: Essential amino acids must be obtained from diet Nonessential amino acids - can be synthesized Nitrogen is needed for amino acids, nucleotides. Atmospheric N2 is the ultimate source of biological nitrogen. Nitrogen fixation: a few bacteria possess nitrogenase which can reduce N2 to ammonia (NH4+). Nitrogen is recycled in nature through the nitrogen cycle.

The Nitrogen cycle The nitrogen cycle. Organic nitrogenous compounds are formed by the incorporation of NH4+ into carbon skeletons. Note that denitrification and nitrogen fixation are anaerobic processes

What is Nitrogenase Complex? Organisms Gain Access to Atmospheric N2 Via the Pathway of Nitrogen Fixation Only occurs in certain prokaryotes Rhizobia fix nitrogen in symbiotic association with leguminous plants. Rhizobia fix N for the plant and plant provides Rhizobia with carbon substrates. All nitrogen fixing systems appear to be identical. They require nitrogenase, a reductant (reduced ferredoxin), ATP, O-free conditions and regulatory controls (ADP inhibits and NH4+ inhibits expression of nif genes) What is Nitrogenase Complex? Two protein components: nitrogenase reductase and nitrogenase Nitrogenase reductase is a 60 kD homodimer with a single 4Fe-4S cluster. It binds MgATP 4ATP required per pair of electrons transferred. Reduction of N2 to 2NH3 + H2 requires 4 pairs of electrons, so 16 ATP are consumed per N2. Nitrogenase is very oxygen-sensitive . Each molecule of enzyme contains 2 Mo, 32 Fe, 30 equivalents of acid-labile sulfide (FeS clusters, etc)

Nitrogenase An enzyme present in Rhizobium bacteria that live in root nodules of leguminous plants. Some free-living soil and aquatic bacteria also possess nitrogenase. Nitrogenase reaction: N2 + 8 H+ + 8 e- + 16 ATP 2 NH3 + H2 + 16 ADP + 16 Pi Ammonia generated from N2 is assimilated into low molecular weight metabolites such as glutamate or glutamine. At pH 7 ammonium ion predominates (NH4+). A second important route in assimilation of ammonia is via glutamine synthetase

Regulation of Nitrogen Fixation ADP inhibits nitrogenase activity; NH4+ represses nif gene expression. In some organisms, the nitrogenase complex is regulated by covalent modification. ADP-ribosylation of nitrogenase reductase leads to its inactivation.

What is the Metabolic Fate of Ammonium? Three major reactions in all cells 1.Carbamoyl-phosphate synthetase I Ammonium is converted to carbamoyl-P two ATP required - one to activate bicarbonate, one to phosphorylate carbamate 2.Glutamate dehydrogenase reductive amination of α-ketoglutarate to form glutamate 3.Glutamine synthetase ATP-dependent amidation of γ-carboxyl of glutamate to glutamine

1. The Carbamoyl-Phosphate Synthetase Reaction is: NH4+ + HCO3- + 2ATP → H2N-COO-PO32- + 2ADP + Pi + 2H+ This reaction is an early step in the urea cycle Note the name “synthetase”, which is reserved for synthetic enzymes that use ATP. Enzymes that synthesize but do not use ATP are termed “synthases”.

2. Glutamate Dehydrogenase The glutamate dehydrogenase reaction.

Figure 3. The glutamine synthetase reaction. The reaction proceeds by activation of the γ-carboxyl group of Glu by ATP, followed by amidation by NH4+.

The Major Pathways of Ammonium Assimilation Lead to Glutamine Synthesis The GDH/GS pathway of ammonium assimilation. The sum of these reactions is the conversion of 1 α-ketoglutarate to 1 glutamine at the expense of 2 NH4+, 1 ATP, and 1 NADPH.

What Regulatory Mechanisms Act on Escherichia coli Glutamine Synthetase? A Case Study in Regulation GS in E. coli 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 But no such regulation occurs in eukaryotic versions of GS

The allosteric regulation of glutamine synthetase.

How Do Organisms Synthesize Amino Acids? Plants and microorganisms can make all 20 amino acids and all other needed N metabolites In these organisms, glutamate is the source of N, via transamination (aminotransferase) reactions Mammals can make only 10 of the 20 amino acids The others are classed as "essential" amino acids and must be obtained in the diet All amino acids are grouped into families according to the intermediates that they are made of.

Humans Can Synthesize Only 10 of the 20 Common Amino Acids

The Difference Between Essential and Nonessential Amino Acids

Amino Groups for Amino Acids Are Derived From Glutamate in Transamination Rxns Glutamate-dependent transamination of α-keto acid carbon skeletons is a primary mechanism for amino acid synthesis. The transamination of oxaloacetate by glutamate to yield aspartate and α-ketoglutarate is a prime example.

The Urea Cycle Acts to Excrete Excess N Through Arg Breakdown N and C in the guanidino group (next slide) of Arg come from NH4+, HCO3- (carbamoyl-P), and the -NH2 of Glu and Asp. Breakdown of Arg in the urea cycle releases two N and one C as urea. Important N excretion mechanism in livers of terrestrial vertebrates. Urea cycle is linked to TCA by fumarate.

The urea cycle series of reactions: The enzymes are (1) ornithine transcarbamoylase (OTCase), (2a and 2b) argininosuccinase synthetase, (3) argininosuccinase, and (4) arginase.

The Urea Cycle Acts to Excrete Excess N Through Arg Breakdown The urea cycle series of reactions: The enzymes are (1) ornithine transcarbamoylase (OTCase), (2a and 2b) argininosuccinase synthetase, (3) argininosuccinase, and (4) arginase.

Questions regarding urea cycle 1. Name 5 -amino acids that are required in the urea cycle. There are 3 traditional, arginine, aspartate, and glutamate, as well 2 non-traditional: ornithine and citrulline. By traditional is meant amino acids that appear in proteins. 2. What is the function of glutamate in the cycle? Glutamate is needed to regenerate aspartate from OAA. 3. All told, how may ATPs are needed to make one molecule of urea? Three are needed . Two to make carbamoyl-PO4 and one to provide energy for the aspartate condensation with citrulline. 4. Why is the urea cycle referred to as a “bicycle”? There are actually 2 cycles going on. One takes ornithine to arginine and returns arginine to ornithine. The second takes fumarate from the argininosuccinate and returns it to aspartate.

Asparagine and Leukemia Leukemia is a cancer of the bone marrow that affects production of lymphocytes. Both normal and malignant lymphocytes are highly dependent on Asparagine uptake from blood. Administration of asparaginase is one therapeutic approach to treat childhood leukemia. Inhibition of asparagine synthetase presents an alternative way to deprive malignant lymphocytes. Adenylated sulfoximines inhibit asparagine synthetase at low concentrations.

Serine Biosynthesis 3-Phosphoglycerate dehydrogenase, an NAD+-dependent enzyme, diverts 3-phosphoglycerate from glycolysis, yielding 3-phosphohydroxpyruvate. Biosynthesis of serine from 3-phosphoglycerate.

Serine Biosynthesis Transamination of 3-phosphohydroxypyruvate by glutamate gives 3-phosphoserine. Serine phosphatase then generates serine. Biosynthesis of serine from 3-phosphoglycerate.

The Aromatic Amino Acids Are Synthesized From Chorismate Shikimate pathway yields chorismate, thence Phe, Tyr, Trp Note the role of chorismate as a branch point in this pathway . His synthesis, like that of Trp, shares metabolic intermediates with purine biosynthetic pathway.

Some of the aromatic compounds derived from chorismate.

Phe, Tyr, and Tryp Are Synthesized From Chorismate The biosynthesis of phenylalanine, tyrosine, and tryptophan from chorismate.

Phe, Tyr, and Tryp Are Synthesized From Chorismate The biosynthesis of phenylalanine, tyrosine, and tryptophan from chorismate.

Phe, Tyr, and Tryp Are Synthesized From Chorismate The biosynthesis of phenylalanine, tyrosine, and tryptophan from chorismate.

Tyrosine is Made From Phenylalanine The formation of tyrosine from phenylalanine.

Amino Acid Biosynthesis Inhibitors as Herbicides A variety of herbicides have been developed as inhibitors of plant enzymes that synthesize “essential” amino acids. These substances show no effect on animals. For example, glyphosate, sold as RoundUp, is a PEP analog that acts as an uncompetitive inhibitor of 3-enolpyruvylshikimate-5-P synthase.

Amino Acid Biosynthesis Inhibitors as Herbicides Inhibitors that act specifically on plant enzymes involved in the synthesis of “essential” amino acids. These substances show no effect on animals.

Histidine Biosynthesis Histidine biosynthesis and purine biosynthesis are connected by common intermediates

How Does Amino Acid Catabolism Lead into Pathways of Energy Production The 20 common amino acids are degraded by 20 different pathways to produce just 7 metabolic intermediates . Metabolic degradation of the common amino acids. Glucogenic amino acids are shown in pink, ketogenic in blue.

Phenylalanine and Tyrosine Are Degraded to Acetoacetate and Fumarate The first reaction in phenylalanine degradation is the hydroxylation reaction of tyrosine biosynthesis Both these amino acids thus share a common degradative pathway Transamination of Tyr yields p-hydroxyphenylpyruvate A vitamin C-dependent dioxygenase then produces homogentisate Ring opening and isomerization gives 4-fumaryl-acetoacetate, which is hydrolyzed to acetoacetate and fumarate