Principles of Biochemistry Horton • Moran • Scrimgeour • Perry • Rawn Principles of Biochemistry Fourth Edition Chapter 17 Amino Acid Metabolism Copyright © 2006 Pearson Prentice Hall, Inc.

Chapter 17 - Amino Acid Metabolism 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

17.1 The Nitrogen Cycle and Nitrogen Fixation 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 Nitrogen is recycled in nature through the nitrogen cycle

Fig 17.1 The Nitrogen cycle Figure 17.1 Nitrogen cycle. A few free-living or symbiotic microorganisms can convert N2 to ammonia. Ammonia is incorporated into biomolecules such as amino acids and proteins, which later are degraded, re-forming ammonia. Many soil bacteria and plants can carry out the reduction of nitrate to ammonia via nitrite. Several bacteria convert ammonia to nitrite. Others oxidize nitrite to nitrate, and some can reduce nitrate to N2.

Nitrogen Fixation Most green plants and some microorganisms contain nitrate reductase and nitrite reductase, enzymes that together catalyze the reduction of nitrogen oxides to ammonina.

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 ATP + 16 Pi

Figure 17.3 Structure of the MoFe7S9N • homocitrate reactive center in Azotobacter vinelandii. (a) Resting state. (b) One possible structure with bound N2. [PDB 2MIN]

Figure 17.3 Structure of the MoFe7S9N • homocitrate reactive center in Azotobacter vinelandii. (a) Resting state. (b) One possible structure with bound N2. [PDB 2MIN]

Figure 17.3 Structure of the MoFe7S9N • homocitrate reactive center in Azotobacter vinelandii. (a) Resting state. (b) One possible structure with bound N2. [PDB 2MIN]

17.2 Assimilation of Ammonia Ammonia generated from N2 is assimilated into low molecular weight metabolites such as glutamate or glutamine At pH 7 ammonium ion predominates (NH4+) At enzyme reactive centers unprotonated NH3 is the nucleophilic reactive species

A. Ammonia Is Incorporated into Glutamate Reductive amination of a-ketoglutarate by glutamate dehydrogenase occurs in plants, animals and microorganisms Glutamine is a nitrogen donor in many biosynthetic reactions

Fig 17.5 Glutamate synthase catalyze the reductive amination of a-ketoglutarate Animals do not have glutamate synthase.

B. Transamination Reactions Transfer of an amino group from an a-amino acid to an a-keto acid In amino acid biosynthesis, the amino group of glutamate is transferred to various a-keto acids generating a-amino acids In amino acid catabolism, transamination reactions generate glutamate or aspartate

Fig 17.6 Transfer of an amino group from an a-amino acid to an a-keto acid

Fig 17.7 Pig (Sus scrofa) cytosolic aspartate transaminase (Space-filling model: the coenzyme pyridoxal phosphate)

Fig 17.8 Assimilation of ammonia into amino acids a. The glutamate dehydrogenase pathway.

Fig 17.8 Assimilation of ammonia into amino acids b. Combined action of glutamine synthetase and glutamate synthase under conditions of low NH4+ concentration.

17.3 Synthesis of Amino Acids Most bacteria and plants (not mammals) synthesize all 20 common amino acids Nonessential amino acids for mammals are usually derived from intermediates of glycolysis or the citric acid cycle (11 of the 20 a.a.) Amino acids with the largest energy requirements are usually essential amino acids

Box 17.3 Essential and Nonessential Amino Acids in Animals

Fig 17.9 Biosynthesis of Amino Acids

A. Asparate and Asparagine Oxaloacetate is the amino-group acceptor in a transamination reaction that produces asparate.

B. Lysine, Methionine, and Threonine Aspartate is the precursor of lysine, methionine, and threonine.

C. Alanine, Valine, Leucine, and Isoleucine Pyruvate is the amino group acceptor in the synthesis of alanine by a transamination reaction.

C. Alanine, Valine, Leucine, and Isoleucine Pyruvate is also a precursor in the synthesis of the branched-chain amino acids valine, leucine, and isoleucine.

D. Glutamate, Glutamine, Arginine, and Proline Fig 17.13 Conversion of glutamate to proline and arginine

E. Serine, Glycine, and Cysteine Serine, glycine, and cysteine- are derived from the glycolytic/gluconeogenic intermediate 3-phosphoglycerate. Fig 17.14 Biosynthesis of serine.

E. Serine, Glycine, and Cysteine Serine, glycine, and cysteine- are derived from the glycolytic/gluconeogenic intermediate 3-phosphoglycerate. Fig 17.15 Biosynthesis of glycine.

E. Serine, Glycine, and Cysteine Serine, glycine, and cysteine- are derived from the glycolytic/gluconeogenic intermediate 3-phosphoglycerate. Fig 17.16 Biosynthesis of cysteine from serine in many bactera and plants.

Fig 17.17 Biosynthesis of cysteine in mammals Animals do not have the normal cysteine biosynthesis pathway shown in fig 17.16.

F. Phenylalanine, Tyrosine, and Tryptophan Chorismate, a derivative of shikimate, is a key branch-point intermediate in aromatic amino acid synthesis. Animals can not synthesize chorismate.

Fig 17.19 Biosynthesis of phenylalanine and tyrosine from chorismate in E. coli

Indole glycerol phosphate for Trp biosynthesis Anthranilate is produced from chorismate Anthranilate is then converted into indole glycerol phosphate for Trp synthesis

Fig 17.21 Reactions catalyzed by tryptophan synthase

G. Histidine

17.4 Amino Acids as Metabolic Precursors The primary role of amino acids is to serve as substances for protein synthesis. Some amino acids are essential precursors in other biosynthesis pathways. Glutamate, glutamine, and asparate Required in the urea cycle Involved in many transamination Purine and pyrimidine biosynthesis Serine and glycine (Fig 17.23) Arginine (Fig 17.24)

Fig 17.23 Compounds formed from serine and glycine

Synthesis of Nitric Oxide (NO) from Arginine Nitric oxide (.N=O) is a gas which can diffuse rapidly into cells, and is a messenger that activates guanylyl cyclase (GMP synthesis) NO relaxes blood vessels, lowers blood pressure, and is a neurotransmitter in the brain (high levels of NO during a stroke kill neurons) Nitroglycerin is converted to NO and dilates coronary arteries in treating angina pectoris

Fig 17.24 Conversion of arginine to nitric oxide and citrulline

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17.5 Protein Turnover Proteins are continuously synthesized and degraded (turnover) (half-lives minutes to weeks) Lysosomal hydrolysis degrades some proteins Some proteins are targeted for degradation by a covalent attachment (through lysine residues) of ubiquitin (C terminus) Proteasome hydrolyzes ubiquitinated proteins

Fig 17.25 Ubiquitination and hydrolysis of a protein Ubiquination enzymes attach multiple ubiquitins Proteasome hydrolyzes uniquinated proteins

17.6 Amino Acid Catabolism Amino acids from degraded proteins or from diet can be used for the biosynthesis of new proteins During starvation proteins are degraded to amino acids to support glucose formation First step is often removal of the a-amino group Carbon chains are altered for entry into central pathways of carbon metabolism

Catabolism of the Carbon Chains of Amino Acids After removal of amino groups, carbon chains of the 20 amino acids can be degraded Degradation products: Citric acid cycle intermediates Pyruvate Acetyl CoA or acetoacetate

Catabolism of Carbon Skeletons Fig 17.26 (next slide) Conversion of the carbon skeletons of amino acids to: Pyruvate Acetoacetate Acetyl CoA Citric acid cycle intermediates

Glucogenic vs Ketogenic Amino Acids Glucogenic amino acids can supply gluconeogenesis pathway via pyruvate or citric acid cycle intermediates Ketogenic amino acids can contribute to synthesis of fatty acids or ketone bodies Some amino acids are both glucogenic and ketogenic

A. Alanine, Asparagine, Aspartate, Glutamate, and Glutamine Reentry into pathways from which carbon skeletons arose by reverse transamination Alanine pyruvate Aspartate oxaloacetate Glutamate a-ketoglutarate Glutamine and asparagine are first hydrolyzed to glutamate and aspartate

B. Arginine, Histidine, and Proline Fig. 17.27

C. Glycine and Serine

Alternate routes for the degradation of threonine to glycine D. Threonine Alternate routes for the degradation of threonine to glycine Figure 17.29 (next slide)

Fig 17.29 Alternate routes for the degradation of threonine

E. The Branched-Chain Amino Acids Leucine, valine and isoleucine are degraded by related pathways The same three enzymes catalyze the first three steps in all pathways A branched-chain amino acid transaminase catalyzes the first step

Fig 17.30 Catabolism of branched-chain amino acids

F. Methionine Fig 17.31 (X represents any of a number of methyl-group acceptors)

F. Methionine Fig 17.31 (X represents any of a number of methyl-group acceptors)

Fig 17.31 (cont)

Fig 17.31 (cont)

G. Cysteine Fig 17.32 Conversion of cysteine to pyruvate

H. Phenylalanine, Tryptophan, and Tyrosine Fig 17.33

Fig 17.34 Conversion of tryptophan to alanine and acetyl CoA

I. Lysine Fig 17.35

I. Lysine Fig 17.35

Fig 17.35 (cont)

17.7 The Urea Cycle Converts Ammonia into Urea Waste nitrogen must be removed (ammonia is toxic to plants and animals) Terrestrial vertebrates synthesize urea (excreted by the kidneys) Birds, reptiles synthesize uric acid

Fig 17.37 A. Synthesis of Carbamoyl Phosphate Synthesis of carbamoyl phosphate (removal of NH3) Catalyzed by carbamoyl phosphate synthetase I (CPS I)

Fig 17.37 Synthesis of carbamoyl phosphate (removal of NH3) catalyzed by carbamoyl phosphate synthetase I (CPS I)

Fig 17.37 (cont)

B. The Reactions of the Urea Cycle Urea cycle (Fig 17.38 & 39 next four slides) Rxn 1 (mitochondria), Rxns 2,3,4 (cytosol) Two transport proteins are required: Citrulline-ornithine exchanger Glutamate-aspartate exchanger Overall reaction for urea synthesis is: NH3 + HCO3- + Aspartate + 3 ADP Urea + Fumarate + 2 ADP + 2 Pi + AMP + PPi

The Urea Cycle

C. Ancillary Reactions of the Urea Cycle Supply of nitrogen for the urea cycle can be balanced by supply of NH3 and amino acids Glutamate dehydrogenase and aspartate transaminase catalyze near equilibrium reactions Flux through these enzymes depends upon relative amounts of ammonia and amino acids Two cases (next slides): (a) NH3 in excess, (b) aspartate in excess

Fig 17.40 Balancing the supply of nitrogen for the urea cycle

NH3 in extreme excess

Aspartate in extreme excess

Glucose-alanine cycle Some amino acids are deaminated in muscle Exchange of glucose and alanine between muscle and liver Provides an indirect means for muscle to eliminate nitrogen and replenish its energy supply

Fig 17.41 Glucose-alanine cycle

17.8 Renal Glutamine Metabolism Produces Bicarbonate Bicarbonate can be lost by buffering H+ in blood Bicarbonate can be replenished by glutamine catabolism in the kidneys a-Ketoglutarate (formed from glutamine oxidation) can be further metabolized to yield bicarbonate Glutamine a-ketoglutarate2- + 2 NH4+

Glutamine → → a-ketoglutarate2- + 2 NH4+ 2 a-ketoglutarate2- → → glucose + 4 HCO3- 2C5H10N2O3 + 3O2 + 6H2O → C6H12O6 + 4HCO3- + 4NH4+

Fig 17.42 Loss of bicarbonate as a buffer