Chapter 22: Biosynthesis of Amino Acids, Nucleotides, and Related Molecules

Question 1

• _____ is crucial in the biosynthesis of the nitrogen-containing compounds.
• Each amino acid and each nucleotide is required in relatively _____ amounts
• The metabolic flow through most of these pathways is not nearly as _____ as the biosynthetic flow leading to carbohydrate or fat in animal tissues.
• Because the different amino acids and nucleotides must be made in the correct _____ and at the right time for protein and nucleic acid synthesis, their biosynthetic pathways must be accurately ______ and _____ with each other
• Because amino acids and nucleotides are ______ molecules, their levels must be regulated to maintain ______ balance in the cell.

Answer 1

• Regulation
• small
• great
• ratios, regulated, coordinated
• charged, electrochemical

Question 2

Because soluble, biologically useful nitrogen compounds are generally scarce in natural environments, most organisms maintain strict economy in their use of _____, ______ _____, and _____.

Answer 2

• ammonia
• amino acids
• nucleotides

Question 3

Nitrogen Cycle

Answer 3

• fixation (reduction)
  
  • atmospheric nitrogen, N₂ is reduced by nitrogen-fixing bacteria to yield ammonia, NH₃ or NH₄⁺
  • Only certain bacteria and archaea can do this
• nitrification
  
  • nitrifying soil bacteria/archaea oxidize ammonia to nitrite NO₂^- and then nitrate NO₃^-
  • nearly all ammonia reaching the soil is oxidized to nitrate
• either of the following two pathways can occur
  
  • Plants & bacteria reduce nitrate and nitrite to ammonia
    
    • ammonia is incorporated into amino acids by plants
    • plants are the source of amino acids for animals
    • as organisms die, microbial degradation of their proteins returns ammonia to the soil
  • denitrification
    
    • bacteria reduces NO₃^- to N₂
    • they use NO₃^- as the electron acceptor
    • generates a transmembrane proton gradient to synthesize ATP
  • anammox
    
    • anaerobic ammonia oxidation
    • bacteria convert ammonia and nitrite to N₂
    • 50% to 70% conversion of NH₃-to-N₂

Question 4

nitrate assimilation

Answer 4

• 90% of the NH₄⁺ generated by vascular plants, algae, and microorganisms comes from nitrate assimilation
• nitrate reductase
  
  • reduces NO₃^- to NO₂^-
  • large, soluble protein (Mr 220,000)
  • NADH donates 2 electrons
  • e- flows through —SH groups of cysteine, FAD, a cytochrome, cofactor containing molybdenum, before reducing the substrate NO₃^- to NO₂^-
• nitrite reductase
  
  • reduces NO₂^- to NH₄⁺
  • in the chloroplasts
  • ferredoxin donates 6 electrons, one at a time
    
    • ferredoxin is reduced in the light-dependent reactions of photosynthesis
  • e- pass through a 4S-4Fe center and siroheme before reducing NO₂^- to NH₄⁺
    
    • metallic center in siroheme carries electrons
    • carboxyl groups of siroheme may donate the 8 protons
  • NADPH is the electron donor in nonphotosynthetic microbes

Question 5

Nitrogen Fixation

summary

Answer 5

• first important product of nitrogen fixation is ammonia
• exergonic reaction
• N₂ + 3H₂ → 2NH₃
• ΔG’° = -33.5 kJ/mol
• N≡N triple bond
  
  • very stable, with a bond energy of 930 kJ/mol
  • extremely high activation energy
  • chemically inert
• Industrial nitrogen fixation
  
  • uses the Haber process
  • requires temps of 400 to 500 ºC, nitrogen and hydrogen at pressures of tens of thousands of kilopascals to provide activation energy
• Biological nitrogen fixation
  
  • occurs @ biological temperatures and at 0.8 atm of nitrogen
  • uses ATP to overcome activation energy barrier
  • N₂ + 10H⁺ + 8e- + 16ATP → 2NH₄⁺ + 16ADP + 16P_i + H₂
  • carried out by the highly conserved nitrogenase complex

Question 6

Nitrogen Fixation

nitrogenase complex - structure

Answer 6

• has 2 components: dinitrogenase reductase and dinitrogenase
• ​dinitrogenase reductase
  
  • (Mr 60,000) is a dimer of 2 identical subunits
  • has a single 4Fe-4S redox center between the subunits
  • oxidized and reduced by one electron
  • each subunit has a binding site for ATP/ADP
• dinitrogenase
  
  • Mr 240,000 is an α2β2 tetramer
  • has 2 Fe-containing cofactors that transfer electrons
    
    • P cluster
      
      • two 4Fe-4S centers that share a sulfur
      • making an 8Fe-7S center
    • FeMo cofactor
      
      • 7 Fe atoms, 9 inorganic S atoms, a Cys side chain, one C in the center of the FeS cluster, and a molybdenum atom, with ligands that include three inorganic S atoms, a His side chain, and two oxygen atoms from a molecule of homocitrate
      • a form of nitrogenase that contains vanadium rather than molybdenum
        
        • bacterial species can produce both types
        • primary nitrogen-fixing system under some conditions

Figure 22-3

• The two identical dinitrogenase reductase, each with a 4Fe-4S redox center and binding sites for two ATP are in green
• The two identical dinitrogenase, each with a P cluster (Fe-S center) and an FeMo cofactor are in purple and blue
• ADP is bound in the ATP site, to make the crystal more stable.

Question 7

Nitrogen Fixation

nitrogenase complex - pathway

Answer 7

• carried out by a highly reduced form of dinitrogenase
• equires eight electrons:
  
  • 6 for the reduction of N2
  • 2 to produce one molecule of H2
• Dinitrogenase is reduced by the transfer of electrons from dinitrogenase reductase
  
  • dinitrogenase tetramer has two binding sites for the reductase
  • a reduced reductase molecule binds to the dinitrogenase and transfers a single electron
  • requires the hydrolysis of 2 ATP molecules by reductase
  • then the oxidized reductase dissociates
  • cycle repeats until eight electrons are transferred
  • N₂ + 10H+ + 8e- + 16ATP → 2NH₄+ + 16ADP + 16P_i + H₂
• source of electrons varies: ferredoxin, reduced flavodoxin, pyruvate, etc.

Question 8

Nitrogen Fixation

nitrogenase complex - role of ATP

Answer 8

• ATP contributes
  
  • chemical energy: through hydrolysis of its phosphoanhydride bonds
  • binding energy: through noncovalent interactions that lower the activation energy
• ATP binding/hydrolysis causes conformational changes that help overcome the high activation energy of nitrogen fixation
  
  • binding of 2 ATP molecules to reductase
    
    • shifts the reduction potential from -300 to -420 mV
    • causes conformation of nitrogenase reductase in two regions
    • brings the 4Fe-4S center of the reductase closer to the P cluster of dinitrogenase, 18 Å to 14 Å away, which facilitates electron transfer between the reductase and dinitrogenase
  • ATP molecules are then hydrolyzed just before the actual transfer of one electron to dinitrogenase

Question 9

Nitrogen Fixation

nitrogenase complex - unstable in oxygen

Answer 9

• nitrogenase complex is remarkably unstable in the presence of oxygen
• reductase is inactivated in air, with a half-life of 30 seconds
• dinitrogenase has a half-life of 10 minutes in air
• Free-living bacteria
  
  • live only anaerobically
  • or repress nitrogenase synthesis when oxygen is present
  • or partially uncouple electron transfer from ATP synthesis so that oxygen is burned off as rapidly as it enters the cell
• symbiotic relationship between leguminous plants and the nitrogen-fixing bacteria
  
  • bacteria in root nodules have access to energy from carbs and citric acid cycle intermediates from the plant
  • bacteria are bathed in a solution of the oxygen-binding heme protein leghemoglobin
    
    • produced by the plant
    • heme may be contributed by the bacteria
    • it binds all available oxygen so that it cannot interfere with nitrogen fixation
    • it delivers the oxygen to the bacterial electron-transfer system

Question 10

Nitrogen Fixation

nitrogenase complex - energetically costly

Answer 10

• Nitrogen fixation is energetically costly
  
  • 16 ATP and 8 electron pairs yield only 2 NH₃
• Tightly regulated, so that NH₃ is produced only when needed
  
  • High [ADP] (means low [ATP]) is a strong inhibitor of nitrogenase
  • NH₄⁺ represses the expression of the ≈20 nitrogen fixation (nif) genes
  • Covalent alteration of nitrogenase is also used in some diazotrophs

Question 11

• Reduced nitrogen, in the form NH₄⁺ is assimulated amino into _____ _____ and then into other nitrogen-containing biomolecules
• Two amino acids, ______ and ______ provide the critical entry point
• These same two amino acids play central roles in the ______ of ammonia and amino groups in amino acid oxidation
• ______ is the source of amino groups for most other amino acids, through transamination reactions
• The amide nitrogen of ______ is a source of amino groups in a wide range of biosynthetic processes
• In most types of cells and in extracellular fluids one or both of these amino acids are present at _____ ______ than other amino acids

Answer 11

• amino acids
• glutamate, glutamine
• catabolism
• Glutamate
• glutamine
• higher concentrations

Question 12

Overview of Amino Acid biosynthesis

Answer 12

• PDF pg 922 image of pathway
• PDF pg 923 table of amino acids

Question 13

• All amino acids are derived from intermediates in
• Nitrogen enters these pathways by way of ______ and _____
• The ______ amino acids are not needed in the diet. The remainder, the ______ amino acids, must be obtained from food
• biosynthetic pathways are grouped into six families corresponding to their ______ ______
• _______ is a notable intermediate in several pathways; it is synthesized from ribose 5-phosphate derived from the pentose phosphate pathway and catalyzed by

Answer 13

• glycolysis, the citric acid cycle, or the pentose phosphate pathway
• glutamate, glutamine
• nonessential, essential
• metabolic precursors
• 5-phosphoribosyl-1-pyrophosphate (PRPP), ribose phosphate pyrophosphokinase

Question 14

α-Ketoglutarate → Glutamate, Glutamine, Proline, and Arginine

biosynthetic pathways to glutamate and glutamine

Answer 14

• glutamine synthetase
  
  • found in all organisms
  • has a central role in amino acid metabolism in mammals converting free NH₄⁺, which is toxic, to glutamine for transport in blood
  • catalyzes the reaction of glutamate and NH₄⁺ to yield glutamine
  • takes place in two steps
    
    1. Glutamate + ATP → γ-glutamyl phosphate + ADP
    2. γ-glutamyl phosphate + NH₄⁺ → glutamine + P_i + H⁺
    • Final EQ = Glutamate + NH₄⁺ + ATP → glutamine + ADP + P_i + H⁺
• glutamate synthase, glutamate: oxoglutarate aminotransferase (GOGAT)
  
  • In bacteria and plants
  • produces glutamate from glutamine
  • α-Ketoglutarate, an intermediate of the citric acid cycle, undergoes reductive amination with glutamine as nitrogen donor
  • α-Ketoglutarate + glutamine + NADPH + H⁺ → 2 glutamate + NADP⁺
  • not present in animals
    
    • instead maintain high levels of glutamate by transamination of α-ketoglutarate during amino acid catabolism
  • another pathway (minor) to produce glutamate
    
    • minor
    • L-glutamate dehydrogenase
      
      • catalyzes α-ketoglutarate to NH₄⁺ to produce glutamate
      • enzyme present in all organisms
      • reducing power comes from NADPH
      • in reverse, is one source of NH₄⁺ for urea
    • reaction equilibrium favors the reactants and the Km for NH₄⁺ is so high the reaction makes a small contribution to NH₄⁺ assimilation
• The net reaction of glutamine synthetase and glutamate synthase
  
  • α-ketoglutarate + NH₄⁺ + NADPH + ATP → glutamine + NADP⁺ + ADP + P_i
• PDF pg. 919

Question 15

α-Ketoglutarate → Glutamate, Glutamine, Proline, and Arginine

biosynthetic pathways to glutamate and glutamine

glutamine synthetase - regulation

Answer 15

• regulated both allosterically and by covalent modification
• Regulation results in a decrease in glutamine synthetase activity when glutamine levels are high, and an increase in activity when glutamine levels are low and α-ketoglutarate and ATP

allosterically

• Alanine, glycine, and at least six end products of glutamine metabolism are allosteric inhibitors of the enzyme
• Each inhibitor alone produces only partial inhibition
• all eight together virtually shut down the enzyme, an example of cumulative feedback inhibition
• Alanine and glycine probably serve as indicators of the general status of amino acid metabolism
• PDF pg. 920, figure 22-8

covalent modification

• inhibition by adenylylation
• AMP is added to Tyr³⁹⁷, located near the enzyme’s active site
• increases sensitivity to the allosteric inhibitors
• glutamine synthetase activity decreases as more subunits are adenylylated
• adenylyltransferase
  
  • catalyzes adenylylation and deadenylylation
  • enzymatic cascade that responds to levels of glutamine, α-ketoglutarate, ATP, and P_i
  • modulated by P_II, a regulatory protein
    
    • P_II is regulated by uridylylation at a Tyr residue
    • uridylyltransferase
      
      • catalyzes uridylated & deuridylated P_II
      • inhibited by glutamine and P_i
      • stimulated by α-ketoglutarate and ATP to P_II
    • uridylated P_II (P_II-UMP) stimulates deadenylylation
      
      • mediates activation of gene transcription of glutamine synthetase
    • deuridylated P_II stimulates adenylylation
      
      • decreases transcription

Question 16

α-Ketoglutarate → Glutamate, Glutamine, Proline, and Arginine

biosynthetic pathways to proline

Answer 16

• α-Ketoglutarate → Glutamate → Proline
• Proline is a cyclized derivative of glutamate
• All five carbon atoms of proline arise from glutamate
• Pathway
  
  • ATP reacts with the γ-carboxyl group of glutamate to form an acyl phosphate
  • acyl phosphate is reduced by NADPH or NADH to glutamate γ-semialdehyde
  • glutamate γ-semialdehyde undergoes spontaneous cyclization and is reduced to proline
• Mammalian pathway
  
  • can also be synthesized from arginine obtained from dietary or tissue protein
  • Arginase converts arginine to ornithine and urea
  • ornithine δ-aminotransferase conerts ornithine to glutamate γ-semialdehyde by the enzyme
  • γ-semialdehyde cyclizes to Δ1-pyrroline-5-carboxylate
  • Δ1-pyrroline-5-carboxylate is converted to proline
• PDF pg. 924

Question 17

α-Ketoglutarate → Glutamate, Glutamine, Proline, and Arginine

biosynthetic pathways to Arginine

Answer 17

• α-Ketoglutarate → Glutamate → Arginine
• ornithine and the urea cycle pathway
  
  • in animals
  • synthesized from glutamate via ornithine and the urea cycle
  • When arginine from dietary intake or protein turnover is insufficient
  • Arginase converts arginine to ornithine and urea
  • ornithine δ-aminotransferase synthesizes ornithine instead of glutamate γ-semialdehyde
  • Ornithine is then converted to citrulline and arginine in the urea cycle
• Bacterial pathway
  
  • de novo biosynthetic pathway for ornithine (and thus arginine)
  • includes two additional steps to avoid spontaneous cyclization
    
    • α-amino group of glutamate is blocked by an acetylation requiring acetyl-CoA
    • the acetyl group is removed to yield ornithine
• PDF pg. 924

Question 18

3-Phosphoglycerate → Serine, Glycine, and Cysteine

biosynthetic pathways to Serine

Answer 18

• 3-Phosphoglycerate → Serine
• hydroxyl group of 3-phosphoglycerate is oxidized by a dehydrogenase (using NAD+) to yield 3-phosphohydroxypyruvate
• Transamination from glutamate yields 3-phosphoserine
• phosphoserine phosphatase hydrolyzes 3-phosphoserine to free serine

Question 19

3-Phosphoglycerate → Serine, Glycine, and Cysteine

biosynthetic pathways to Glycine

Answer 19

• 3-Phosphoglycerate → Serine → Glycine
• Pathway
  
  • hydroxymethyltransferase removes a carbon atom from serine (3 carbons) and creates glycine (2 carbons)
  • reversible
  • requires pyridoxal phosphate
• In the vertebrate livers
  
  • can be made by reverse of the reaction shown catalyzed by glycine synthase (glycine cleavage enzyme)

Question 20

3-Phosphoglycerate → Serine, Glycine, and Cysteine

biosynthetic pathways to cysteine

Answer 20

• 3-Phosphoglycerate → Serine → cysteine
• Plants and bacteria produce the reduced sulfur required for the synthesis of cysteine (and methionine) from environmental sulfates
• Pathway
  
  • Sulfate is activated in two steps to produce 3’-phosphoadenosine 5’-phosphosulfate (PAPS)
  • PAPS undergoes an eight-electron reduction to sulfide
  • sulfide used to form cysteine from serine in a two-step pathway.
• Mammalian pathway
  
  • synthesize cysteine from two amino acids:
    
    • methionine furnishes the sulfur atom
    • serine furnishes the carbon skeleton
  • Methionine converted to S-adenosylmethionine
  • S-adenosylmethionine loses its methyl group and forms S-adenosylhomocysteine (adoHcy)
  • adoHcy is hydrolyzed to free homocysteine
  • homocysteine undergoes a reaction with serine, catalyzed by cystathionine β-synthase, to yield cystathionine
  • cystathionine γ-lyase (requires PLP) catalyzes removal of ammonia and cleavage of cystathionine to yield free cysteine.

Question 21

Oxaloacetate → Asparte

Asparte → Asparagine

Asparte → Methionine

Asparte → Lysine

Asparte → Threonine

biosynthetic pathways

Answer 21

• Oxaloacetate → Asparte
• Aspartate is synthesized by transamination from glutamate
• Asparte → Asparagine
  
  • synthesized by amidation of aspartate
  • glutamine donating the NH₄⁺
  • nonessential amino acids
  • have simple biosynthetic pathways that occur in all organism
• Methionine, threonine, lysine
  
  • essential amino acids
  • humans cannot synthesize them
  • biosynthetic pathways are complex and interconnected
  • In some cases, the pathways in bacteria, fungi, and plants differ significantly
  • Pathway keypoints
    
    • Branch points occur at aspartate β-semialdehyde & homoserine
      
      • β-semialdehyde: intermediate in all three pathways
    • homoserine: precursor of threonine and methionine
  • Threonine a precursors of isoleucine

Question 22

Pyruvate → Alanine

Pyruvate → Valine

Pyruvate → Leucine

Pyruvate → Isoleucine

biosynthetic pathways

Answer 22

• Pyruvate → Alanine
  
  • synthesized by transamination from glutamate
  • nonessential amino acids
  • have simple biosynthetic pathways that occur in all organism
• isolecine, valine and leucine
  
  • essential amino acids
  • humans cannot synthesize them
  • biosynthetic pathways are complex and interconnected
  • In some cases, the pathways in bacteria, fungi, and plants differ significantly
  • valine and isoleucine pathways share four enzyme
  • valine pathways
    
    • begin with condensation of two carbons of pyruvate with another molecule of pyruvate
  • isoleucine in pathways
    
    • begin with condensation of two carbons of pyruvate with α-ketobutyrate
      
      • α-ketobutyrate is derived from threonine
      • requires pyridoxal phosphate
  • leucine pathway
    
    • α-ketoisovalerate intermediate, is the starting point for a four-step branch pathway

Question 23

Phosphoenolpyruvate + Erythrose 4-phosphate

→ Phenylalanine, Tyrosine, Tryptophan

Phenylalanine → Tyrosine

biosynthetic pathways

Answer 23

• Aromatic rings not readily available in environment
• Shared/main patway in bacteria, fungi, and plants, is the main biological route of aromatic ring formation
• Proceeds through ring closure of an aliphatic precursor followed by stepwise addition of double bonds
• Pathway
  
  • first four steps produce shikimate
    
    • seven-carbon molecule
    • derived from erythrose 4-phosphate and phosphoenolpyruvate
  • converted to chorismate in three steps
    
    • includes addition of three more carbons from another molecule of phosphoenolpyruvate
  • tryptophan branch
    
    • chorismate is converted to anthranilate
      
      • glutamine donates the nitrogen that will become part of the indole ring
    • Anthranilate condenses with PRPP
      
      • indole ring is derived from the ring carbons and amino group of anthranilate plus two carbons derived from PRPP
      • catalyzed by tryptophan synthase
        
        • has an α2β2 subunit structure and can be dissociated into two α & β subunits that catalyze different parts of the overall reaction
        • some enzyme activities requires a noncovalent association with other enzymes of the pathway
        • enzymes are components of a large, multienzyme complex in both bacteria and eukaryotes
      • requires pyridoxal phosphate
      • Indole formed in the first part is not released by the enzyme, instead moves through a channel from the α to β-subunit active sites (Intermediate channeling)
      • it condenses with a Schiff base intermediate derived from serine and PLP
  • phenylalanine and tyrosine
    
    • In plants and bacteria,
    • synthesized from chorismate
    • pathways much less complex than tryptophan
    • common intermediate is prephenate
    • final step is transamination with glutamate
  • Phenylalanine → Tyrosine
    
    • Animals
    • through hydroxylation at C-4 of the phenyl group by phenylalanine hydroxylase
    • same enzyme participates in the degradation of phenylalanine
    • a conditionally essential amino acid, or as nonessentia cuz it can be synthesized from the essential amino acid phenylalanine.

Notes from pathway

• All carbons are derived from either erythrose 4-phosphate or phosphoenolpyruvate
• NAD+ required as a cofactor in step 2 is released unchanged; it may be transiently reduced to NADH during the reaction
• Step 6 is competitively inhibited by glyphosate (2COOOCH2ONHOCH2OPO223 ), the active
  ingredient in the widely used herbicide Roundup
• PDF pg. 930

Question 24

Ribose 5-phosphate → Histidine

biosynthetic pathways to

Answer 24

• pathway to histidine in all plants and bacteria differs
• derived from three precursors
  
  • PRPP contributes five carbons
  • purine ring of ATP contributes a nitrogen and a carbon
  • glutamine supplies the second ring nitrogen
• key steps are condensation of ATP and PRPP
• use of ATP as a metabolite dovetails with the purine biosynthetic pathway
• remnant of ATP an intermediate of purine biosynthesis that is rapidly recycled to ATP

Question 25

Amino Acid Biosynthesis Allosteric Regulation

Answer 25

• regulation takes place in part through feedback inhibition of the first reaction by the end product of the pathway
• This first reaction is often catalyzed by an allosteric enzyme that plays an important role in the overall control of flux through that pathway
• concerted inhibition
  
  • more complex
  • products derived from end product serve as negative feedback modulators of the enzyme, and the overall effects of these and other modulators are more than additive
• Additional mechanisms contribute to the regulation of the amino acid biosynthetic pathways. Because the 20 common amino acids must be made in the correct proportions for protein synthesis

Question 26

In addition to their role as the building blocks of proteins, amino acids are precursors of many specialized biomolecules, including

Answer 26

hormones, coenzymes, nucleotides, alkaloids, cell wall polymers, porphyrins, antibiotics, pigments, and neurotransmitters

Question 27

Glycine Is a Precursor of Porphyrins

Answer 27

• glycine is a major precursor for the biosynthesis of prophyrins
  
  • central importance of the porphyrin nucleus in heme proteins: hemoglobin and the cytochromes
• porphyrins are constructed from
  
  • four molecules of the monopyrrole derivative porphobilinogen
  • porphobilinogen is derived from two molecules of δ-aminolevulinate.
    
    • two major pathways to δ-aminolevulinate
    • In higher eukaryotes
      
      • glycine reacts with succinylCoA & yields α-amino-β-ketoadipate
      • α-amino-β-ketoadipate decarboxylated to δ-aminolevulinate
  • two molecules of δ-aminolevulinate condense to form porphobilinogen
  • four molecules of porphobilinogen come together to form protoporphyrin
  • iron atom is incorporated catalyzed by ferrochelatase
  • regulated in higher eukaryotes by heme which serves as a feedback inhibitor of early steps
• Genetic defects in the biosynthesis of porphyrins can lead to the accumulation of pathway intermediates, causing a variety of human diseases known collectively as porphyrias
  *

Question 28

• iron-porphyrin (heme) group of hemoglobin, released from dying erythrocytes in the spleen, is degraded to yield free ______ and, ultimately, ______ which is largely insoluble travels.
• heme breakdown play significant roles in protecting cells from _____ _____ and in regulating certain cellular functions
• ______ is the most abundant antioxidant in mammalian tissues

Answer 28

• Fe²⁺, Bilirubin
• oxidative damage
• Bilirubin

Question 29

bilirubin pathways

Answer 29

• bilirubin synthesis
  
  • two-step pathway
  1. heme oxygenase, converts heme to
     
     • biliverdin: a linear tetrapyrrole derivative
     • other products:
       
       • Fe²⁺
         
         • quickly bound by ferritin
       • CO
         
         • binds to hemoglobin
         • 1% of an individual’s heme is complexed with CO
         • have some regulatory and/or signaling functions
         • acts as a vasodilator
         • regulatory effects on neurotransmission
  2. biliverdin reductase converts biliverdin to bilirubin
• Bilirubin pathway
  
  • complexes with serum albumin in bloodstream
  • Liver transforms bilirubin bile pigment: bilirubin diglucuronide
  • bilirubin diglucuronide is secreted into small intestine
  • in small intestine, bilirubin diglucuronide is converted to urobilinogen by microbial enzymes
    
    • Some urobilinogen is reabsorbed into the blood and transported to the kidney
      
      • kidney coverts urobilinogen to urobilin which gives urine its yellow color
    • Urobilinogen remaining in intestine
      
      • converted to stercobilin (gives red-brown color to feces)
• PDF pg. 936

Question 30

heme degradation pathway is subject to regulation

Answer 30

• mainly at the first step
• Humans have at least three isozymes of heme oxygenase (HO)
  
  • HO-1
    
    • highly regulated
    • expression of its gene is induced by a wide range of stress conditions (shear stress, angiogenesis (uncontrolled development of blood vessels), hypoxia, hyperoxia, heat shock, exposure to ultraviolet light, hydrogen peroxide, and many other metabolic insults).
  • HO-2
    
    • found mainly in brain and testes
    • continuously expressed
  • HO-3
    
    • not yet well characterized

Question 32

Precursor of Creatine and Glutathione

Answer 32

• Creatine
  • synthesized from glycine and arginine
  • methionine, in the form of S-adenosylmethionine, acts as methyl group donor
  • Phosphocreatine
    
    • derived from creatine
    • energy buffer in skeletal muscle
• Glutathione (GSH)
  • redox buffer
  • derived from glutamate, cysteine, and glycine
  • catalized glutathione peroxidase
  • γ-carboxyl group of glutamate is activated by ATP & forms acyl phosphate intermediate
  • oxidized form of glutathione contains two glutathione molecules linked by a disulfide bond
  • helps maintain the sulfhydryl groups of proteins in the reduced state and iron of heme in the ferrous (Fe²⁺) state
  • reducing agent for glutaredoxin
  • removes toxic peroxides

Question 33

• _____ acids do not generally occur in proteins
• They are found primarily in bacteria, in the structure of bacterial _____ ______ and ______ ______
• they arisedirectly from the _____ _____

Answer 33

• D-amino
• cell walls, peptide antibiotics
• L isomers

Question 34

Aromatic Amino Acids Are Precursors of Many Plant Substances

Answer 34

• Phenylalanine and tyrosine
  
  • lignin
    
    • rigid polymer
    • second only to cellulose in abundance in plant tissues
  • tannins that inhibit oxidation in wines
  • alkaloids such as morphine
  • flavoring of cinnamon oil, nutmeg, cloves, vanilla, cayenne pepper, and other products
• Tryptophan
  
  • auxin
    
    • plant growth hormone indole-3acetate
    • Tryptophan

Question 35

Precursors of Neurotransmitters

Answer 35

• dopamine, norepinephrine, and epinephrine
  
  • a family of catecholamines
  • derived from tyrosine
• γ-aminobutyrate (GABA)
  
  • inhibitory neurotransmitter
  • underproduction associated with epileptic seizures.
  • derived from Glutamate decarboxylation
• serotonin
  
  • tryptophan
• histamine
  
  • powerful vasodilator in animal tissues
  • released in large amounts as part of allergic response
  • stimulates acid secretion in the stomach
  • derived from histidine which undergoes decarboxylation
• spermine and spermidine
  
  • Polyamines
  • involved in DNA packaging
  • derived from methionine and ornithine

Question 36

Precursor for Nitric Oxide

Answer 36

• biological messenger
• synthesized from arginine
• NADPH-dependent reaction catalyzed by nitric oxide synthase
  
  • dimeric enzyme structurally related to NADPH cytochrome P-450 reductase
  • five-electron oxidation
  • Each subunit contains one bound molecule of each of four different cofactors: FMN, FAD, tetrahydrobiopterin, and Fe³⁺ heme.
• unstable molecule
• cannot be stored
• synthesis stimulated by interaction of nitric oxide synthase with Ca2calmodulin

Question 37

De Novo and Salvage Pathway Synthesis

Summary

Answer 37

• De Novo
  
  • begins with precursors: amino acids, ribose 5-phosphate, CO2, and NH3
  • identical in all living organisms
  • free bases guanine, adenine, thymine, cytidine, and uracil are not intermediates
    
    • not synthesized and then attached to ribose
  • precursor: Phosphoribosyl pyrophosphate (PRPP)
  • structure of ribose is retained
  • Glutamine most important source of amino groups
  • purine ring
    
    • built one or a few atoms at a time
    • attached to ribose throughout the process
    • precursor: glycine
    • Aspartate used as source of an amino group
  • pyrimidine ring
    
    • synthesized as orotate
    • attached to ribose phosphate
    • converted to pyrimidine nucleotide
    • precursor: aspartate
• Salvage
  
  • recycle the free bases and nucleosides released from nucleic acid breakdown
  • free bases are intermediates in some of the salvage pathways
• enzymes are present as large, multienzyme complexes in the cell
• cellular pools of nucleotides (other than ATP) are quite small
  
  • <= 1% amounts required to synthesize cell’s DNA
• nucleotide synthesis may limit the rates of DNA replication and transcription

Question 38

De Novo Purine Nucleotide Synthesis

Answer 38

• two parent purine nucleotides of nucleic acids are
  
  • adenosine 59-monophosphate (AMP; adenylate)
    
    • contains adenine
  • guanosine 59-monophosphate (GMP; guanylate)
    
    • contains guanine
• Pathway
  
  1. amino group donated by glutamine
     
     • attached at C-1 of PRPP
     • forms 5-phosphoribosylamine
       
       • highly unstable
       • half-life of 30 seconds at pH 7.5
       • purine ring is derived from this structure
  2. addition of three atoms from glycine
     
     • condensation reaction
     • ATP consumed to activate glycine carboxyl group (in the form of an acyl phosphate)
  3. added glycine amino group is formylated by N¹⁰-formyltetrahydrofolate
  4. nitrogen contributed by glutamine
  5. dehydration and ring closure yield 5-aminoimidazole ribonucleotide
     
     • five-membered imidazole ring of the purine nucleus
     • AIR step
  6. carboxylation
     
     • bacteria / fungi
       
       • carboxyl group added
         
         • 3 of the 6 atoms needed for the second ring in purine are already in place.
       • rearrangement transfers carboxylate from exocyclic amino group to position 4 of imidazole ring
         
         • uses bicarbonate instead of biotin
     • higher eukaryotes
       
       • carboxylated directly to carboxyaminoimidazole ribonucleotide by AIR carboxylase
  7. (see 2nd bullet of bacteria)
  8. Aspartate now donates its amino group and forms an amide bond
  9. Aspartate’s cabon skeleton is eliminated
     
     • similar to urea cycle
  10. final carbon is contributed by N¹⁰-formyltetrahydrofolate
  11. second ring closure takes place
      
      • yields second fused ring of purine nucleus
• first intermediate with a complete purine ring is inosinate (IMP)
  
  • Conversion of inosinate to adenylate requires insertion of an amino group derived from aspartate
  • GTP rather than ATP is the source of the high-energy
• steps 1, 3, and 5 in are catalyzed by a multifunctional protein

Question 39

Three major feedback mechanisms cooperate in regulating the

• overall rate of de novo purine nucleotide synthesis
• relative rates of formation of the two end products, adenylate and guanylate

Answer 39

1. exerted on the first reaction that is unique to purine
   
   • transfer of an amino group to PRPP to form 5-phosphoribosylamine
   • catalyzed by allosteric enzyme glutamine-PRPP amidotransferase
   • inhibited by end products IMP, AMP, and GMP
   • AMP and GMP act synergistically
     
     • whenever AMP or GMP accumulates to excess, the first step in its biosynthesis from PRPP is partially inhibited
2. excess of GMP in the cell inhibits formation of xanthylate from inosinate by IMP dehydrogenase; does not affect the formation of AMP
   
   • Conversely, an accumulation of adenylate inhibits formation of adenylosuccinate by adenylosuccinate synthetase, without affecting the biosynthesis of GMP
   • When both are present in sufficient quantity, IMP builds up and inhibits an earlier step
3. GTP is required in conversion of IMP to AMP. Whereas ATP is required for conversion of IMP to GMP
4. inhibition of PRPP synthesis by allosteric regulation of ribose phosphate pyrophosphokinase
   
   • this enzyme is inhibited by ADP and GDP, in addition to metabolites

Question 40

De Novo Pyrimidine Nucleotide Synthesis

Answer 40

• common pyrimidine ribonucleotides
  
  • cytidine 59-monophosphate (CMP; cytidylate)
    
    • contain cytosine
  • uridine 59-monophosphate (UMP; uridylate
    
    • contain uracil
• six-membered pyrimidine ring is made first and then attached to ribose 5-phosphate
• carbamoyl phosphate is required
  
  • intermediate in the urea cycle
  • humans
    
    • made in the cytosol by carbamoyl phosphate synthetase II
  • bacteria
    
    • one enzyme supplies carbamoyl phosphate for the synthesis of arginine and pyrimidines
• reacts with aspartate to yield
  N-carbamoylaspartate, catalyzed by aspartate transcarbamoylase​
  
  • step higly regulated in bacteria
• dihydroorotase removes water from N-carbamoylaspartate
  
  • closes pyrimidine ring
  • forms L-dihydroorotate
• L-dihydroorotate is oxidized to the pyrimidine derivative orotate
  
  • NAD+ is the ultimate electron acceptor
• In eukaryotes, first three enzymes in pathway (carbamoyl phosphate synthetase II, aspartate transcarbamoylase, and dihydroorotase) are part of a single trifunctional protein, CAD
  
  • three identical polypeptide chains
  • each with active sites for all three reactions
• ribose 5-phosphate side chain is attached to orotate by PRPP to yield orotidylate
• orotidylate is decarboxylated to uridylate
• uridylate is phosphorylated to UTP
• CTP is formed from UTP by cytidylate synthetase
  
  • consumes one ATP
  • nitrogen donor is normally glutamine

Question 41

Pyrimidine Nucleotide Biosynthesis Is Regulated by

Feedback Inhibition

Answer 41

• In bacteria it’s done via aspartate transcarbamoylase (ATCase)
• catalyzes the first reaction in the sequence and is inhibited by CTP, the end product
• exists in two conformations, active and inactive
• active
  
  • CTP is not bound to the regulatory subunits
  • enzyme is maximally active
• inactive
  
  • CTP accumulates and binds
  • changes conformation which is transmitted to catalytic subunits and shift to an inactive conformation
• ATP prevents the changes induced by CTP

Question 42

Nucleotides are converted to nucleoside triphosphates

Answer 42

• conversion pathways are common to all cells
• adenylate kinase
  
  • phosphorylates AMP to ADP
  • ATP + AMP ⇔ 2 ADP
• nucleoside monophosphate kinases
  
  • allows ATP to generate other nucleoside diphosphates
  • specific for a particular base but nonspecific for the sugar
  • ATP + NMP ⇔ ADP + NDP
• nucleoside diphosphate kinase
  
  • converts nucleoside diphosphates to triphosphates
  • is not specific for the base (purines or pyrimidines) or the sugar (ribose or deoxyribose)
  • NTP_D + NDP_A ⇔ ND_D + NTP_A

Question 43

Ribonucleotides Are the Precursors of Deoxyribonucleotides

Answer 43

• Deoxyribonucleotides, the building blocks of DNA, are derived from the corresponding ribonucleotides
• ribonucleotide reductase
  
  • catalyses Reduction occurs at the 29-carbon atom of the D-ribose to form the 29-deoxy derivative
  • reduction occurs at a nonactivated carbon
  • reduces nucleoside diphosphates (NDPs) to deoxyribonucleoside diphosphates (dNDPs)
  • requires a pair of hydrogen atoms
  • involves free radicals in biochemical transformations
• thioredoxin
  
  • intermediate hydrogen-carrying protein
  • helps NADPH donate pair of hydrogen atoms to ribonucleotide reductase
  • ribonucleotide reductase uses reduced thioredoxin
  • has pairs of —SH groups that carry hydrogen atoms from NADPH to ribonucleoside diphosphate
  • thioredoxin reductase reduces oxidized (disulfide) form via NADPH
• glutaredoxin
  
  • second source of reducing equivalents is glutathione (GSH), the reductant for glutaredoxin
  • glutaredoxin transfers reducing power to ribonucleotide reductase

Question 44

ribonucleotide reductase structure

Answer 44

• α2β2 dimer, with catalytic subunits α2 and radical-generation subunits β2
• catalytic subunit contains two kinds of regulatory sites
• formed at the interface between the catalytic and radical-generation
• α2 subunit
  
  • contributes two sulfhydryl groups
• β2 subunits
  
  • contribute a stable tyrosyl radical
  • has binuclear iron (Fe³⁺) cofactor that helps generate and stabilize the Tyr122 radical
  • tyr122 too far from active site
    
    • several aromatic residues form a long-range radical transfer pathway to active site
• three classes
  
  • differ in identity of the group supplying the active-site radical and in cofactors used to generate itclass I)
    
    • requires oxygen to regenerate the tyrosyl radical
    • functions only in aerobic environment
  • class II
    
    • has 59-deoxyadenosylcobalamin instead of binuclear iron center
  • Class III
    
    • anaerobic environment
• regulation
  
  • activity & substrate specificity is regulated by the binding of effector molecules
  • α2 has two types of regulatory sites
    
    • One type
      
      • affects overall enzyme activity
      • binds either ATP, which activates the enzyme, or dATP, which inactivates it
    • second type
      
      • alters substrate specificity in response to effector molecule—ATP, dATP, dTTP, or dGTP—that is bound there
      • ATP or dATP bound → reduction of UDP and CDP favored
      • dTTP or dGTP bound → reduction of GDP or ADP stimulated
  • ATP is a general activator for biosynthesis and ribonucleotide reduction
    
    • dATP in small amounts → increases reduction of pyrimidine nucleotides
    • oversupply of pyrimidine dNTPs, signaled by high levels of dTTP → reduction of GDP
    • High levels of dGTP → ADP reduction
    • high levels of dATP → shut enzyme down
  • regulatory effects come with large structural rearrangements in the enzyme
    
    • residues in the path are exposed to solvent preventing radical transfer inhibiting the reaction
    • reversed when dATP levels are reduced

Question 45

thymine de novo pathway

Answer 45

• involves only deoxyribonucleotides
• immediate precursor of thymidylate (dTMP) is dUMP
• In bacteria
  
  • pathway to dUMP begins with formation of dUTP, by deamination of dCTP or by phosphorylation of dUDP
  • dUTP is converted to dUMP by a dUTPase
• thymidylate synthase catalyzes dUMP to dTMP
• A one-carbon unit at the hydroxymethyl (—CH2OH) oxidation level is transferred from N5,N10-methylenetetrahydrofolate to dUMP, then reduced to a methyl group
• reduction occurs at the expense of oxidation of tetrahydrofolate to dihydrofolate
• dihydrofolate is reduced to tetrahydrofolate by dihydrofolate reductase

Question 46

Degradation of Purines and Pyrimidines Produces

Uric Acid and Urea, Respectively

Answer 46

• Purine nucleotides degradattion
  
  • they lose their phosphate through the action of 59-nucleotidase
  • Pathway
    
    • Adenylate yields adenosine
    • adenosine is deaminated to inosine by adenosine deaminase
    • inosine is hydrolyzed to hypoxanthine (its purine base) and D-ribose
    • Hypoxanthine is oxidized successively to xanthine and then uric acid by xanthine oxidase
      
      • flavoenzyme with an atom of molybdenum
      • has four iron-sulfur centers in its prosthetic group
      • Molecular oxygen is the electron acceptor
  • Uric acid
    
    • excreted end product of purine catabolism in primates, birds, and some other animals
    • arises in part from ingested purines and turnover of the purine nucleotides of nucleic acids
    • in most mammals uric acid is further degraded to allantoin by the action of urate oxidase
• Pyrimidine nucleotides degradation
  
  • GMP catabolism also yields uric acid as end product.
  • Pathway
    
    • GMP is first hydrolyzed to guanosine
    • guanosine is then cleaved to free guanine
    • Guanine undergoes hydrolytic removal of its amino group to yield xanthine
    • xanthine is converted to uric acid by xanthine oxidase
  • pathways for degradation of pyrimidines generally lead to NH⁺₄ production and urea synthesis
• Page pdf 952

Question 47

Purine and Pyrimidine Bases Are Recycled by Salvage Pathways

Answer 47

• purine and pyrimidine bases are constantly released in cells during the metabolic degradation of nucleotides
• salvaged and reused to make nucleotides
• pathway is much simpler than the de novo synthesis
• One primary salvage pathways
  
  • catalyzed by adenosine phosphoribosyltransferase,
  • free adenine reacts with PRPP to yield adenine nucleotide
  • Free guanine and hypoxanthine (the deamination product of adenine) are salvaged in the same way by hypoxanthine-guanine phosphoribosyltransferase

Question 48

allopurinol

Answer 48

• inhibits xanthine oxidase, the enzyme that catalyzes the conversion of purines to uric acid
• a substrate of xanthine oxidase, which converts allopurinol to oxypurinol (alloxanthine).
• Oxypurinol inactivates the reduced form of the enzyme by remaining tightly bound in its active site
• When xanthine oxidase is inhibited, the excreted products of purine metabolism are xanthine and hypoxanthine

Question 49

Many Chemotherapeutic Agents Target Enzymes in the Nucleotide Biosynthetic Pathways

Answer 49

PDF pg 954