22| Biosynthesis of Amino Acids, Nucleotides, and Related Molecules

22| Biosynthesis of Amino Acids, Nucleotides, and Related Molecules
© 2017 W. H. Freeman and Company

CHAPTER 22 Biosynthesis of Amino Acids, Nucleotides, and Related Molecules
Learning goals: Incorporation of ammonia into biomolecules Biosynthesis of amino acids Biosynthesis of nucleotides Catabolism of purines

Importance of Nitrogen in Biochemistry
Nitrogen (with H, O, and C) is a major elemental constituent of living organisms. Mostly in nucleic acids and proteins But also found in: several cofactors (NAD, FAD, biotin … ) many small hormones (epinephrine) many neurotransmitters (serotonin) many pigments (chlorophyll) many defense chemicals (amanitin) ‘

Ammonia Is Incorporated into Biomolecules Through Glu and Gln
Glutamine is made from Glu by glutamine synthetase in a two-step process. Phosphorylation of Glu creates a good leaving group that can be easily displaced by ammonia. FIGURE 188 Ammonia transport in the form of glutamine. Excess ammonia in tissues is added to glutamate to form glutamine, a process catalyzed byglutamine synthetase. After transport in the bloodstream, the glutamine entersthe liver, and NH+4 is liberated in mitochondria by the enzyme glutaminase.

Structure of Gln Synthetase
FIGURE 22–7 Subunit structure of bacterial type I glutamine synthetase. (PDB ID 2GLS) This view shows 6 of 12 the identical subunits; a second layer of 6 subunits lies directly beneath the six shown. Each of the 12 subunits has an active site, where ATP and glutamate are bound in orientations that favor transfer of a phosphoryl group from ATP to the side-chain carboxyl of glutamate. In this crystal structure, ADP occupies the ATP site.

Adenylation of Glutamine Synthetase
Adenylylation (attachment of AMP) to Tyr-397 assists in inhibition. Increases sensitivity to inhibitors Part of complex cascade that is dependent on [Glu], [-ketoglutarate], [ATP], and [Pi] Activity of adenylyltransferase regulated by binding to regulatory protein PII FIGURE 22–9 Second level of regulation of glutamine synthetase: covalent modifications. (a) An adenylylated Tyr residue. (b) Cascade leading to adenylylation (inactivation) of glutamine synthetase. AT represents adenylyltransferase; UT, uridylyltransferase. PII is a regulatory protein, itself regulated by uridylylation. Details of this cascade are discussed in the text.

PII Is Regulated by Uridylylation
(Remember that PII regulates adenylyltransferase, which helps inhibit Gln synthetase.) When PII is uridylylated, adenylyltransferase stimulates deadenylylation of Gln synthetase (increasing the latter’s activity). ALSO, uridylylated PII upregulates transcription of Gln synthetase.

Biosynthesis of Amino Acids and Nucleotides― MultipleTransaminations
Transaminations and rearrangements using pyridoxal phosphate (PLP) PLP is active form of vitamin B6 Catalyzed by amidotransferases PLP has aldehyde group that forms Schiff base with Lys of aminotransferase

Proposed Mechanism for Glutamine Amidotransferases
Two domains: one binds Gln other is amino group acceptor and binds substrate Cys acts as nucleophile to cleave amide bond of Gln Forms glutamyl-enzyme intermediate Then second substrate binds to accept amino group from enzyme MECHANISM FIGURE 22–10 Proposed mechanism for glutamine amidotransferases. Each enzyme has two domains. The glutamine-binding domain contains structural elements conserved among many of these enzymes, including a Cys residue required for activity. The NH3-acceptor (secondsubstrate) domain varies. Two types of amino acceptors are shown. X represents an activating group, typically a phosphoryl group derived from ATP, that facilitates displacement of a hydroxyl group from R—OH by NH3.

Amino Acid Synthesis Overview
Source of N is Glu or Gln Derive from intermediates of: glycolysis citric acid cycle pentose phosphate pathway Bacteria can synthesize all 20. Mammals require some in diet. FIGURE Overview of amino acid biosynthesis. The carbon skeleton precursors derive from three sources: glycolysis (light red), the citric acid cycle (blue), and the pentose phosphate pathway (purple).

All Amino Acids Derive from One of Seven Precursors
CAC: -ketoglutarate, oxaloacetate Glycolysis pyruvate, 3-phosphoglycerate, phosphoenolpyruvate Pentose phosphate pathway ribose 5-phosphate, erythrose 4-phosphate

TABLE 22-1 Amino Acid Biosynthetic Families, Grouped by Metabolic Precursor α-Ketoglutarate Glutamate Glutamine Proline Arginine Pyruvate Alanine Valinea Leucinea Isoleucinea 3-Phosphoglycerate Serine Glycine Cysteine Phosphoenolpyruvate and erythrose 4-phosphate Tryptophana Phenylalaninea Tyrosineb Oxaloacetate Aspartate Asparagine Methioninea Threoninea Lysinea Ribose 5-phosphate Histidinea aEssential amino acids in mammals. bDerived from phenylalanine in mammals.

Proline and Arginine Derive from Glutamate
Glutamate is derived from transamination of α-ketoglutarate, as seen in Chapter 18.

Biosynthesis of Pro and Arg from Glu in Bacteria
ATP reacts with -carboxyl group  acyl phosphate. NADPH or NADH reduces the acyl phosphate to a semialdehyde that rapidly cyclizes. Final reduction step yields proline. FIGURE 22–12 Biosynthesis of proline and arginine from glutamate in bacteria. All five carbon atoms of proline arise from glutamate. In many organisms, glutamate dehydrogenase is unusual in that it uses either NADH or NADPH as a cofactor. The same may be true of other enzymes in these pathways. The γ-semialdehyde in the proline pathway undergoes a rapid, reversible cyclization to ∆1-pyrroline-5-carboxylate (P5C), with the equilibrium favoring P5C formation. Cyclization is averted in the ornithine/arginine pathway by acetylation of the α-amino group of glutamate in the first step and removal of the acetyl group after the transamination. Although some bacteria lack arginase and thus the complete urea cycle, they can synthesize arginine from ornithine in steps that parallel the mammalian urea cycle, with citrulline and argininosuccinate as intermediates (see Fig. 18–10). Here, and in subsequent figures in this chapter, the reaction arrows indicate the linear path to the final products, without considering the reversibility of individual steps. For example, the step of the pathway leading to arginine that is catalyzed by N-acetylglutamate dehydrogenase is chemically similar to the glyceraldehyde 3-phosphate dehydrogenase reaction in glycolysis (see Fig. 14–8), and is readily reversible.

In Animals, Proline Can ALSO Be Synthesized from Arginine
Ornithine is derived from the urea cycle or degradation of arginine. Ornithine -aminotransferase converts ornithine to glutamate -semialdehyde that cyclizes and converts to Pro. FIGURE 22–13 Ornithine δ-aminotransferase reaction: a step in the mammalian pathway to proline. This enzyme is found in the mitochondrial matrix of most tissues. Although the equilibrium favors P5C formation, the reverse reaction is the only mammalian pathway for synthesis of ornithine (and thus arginine) when arginine levels are insufficient for protein synthesis.

Arginine Is Synthesized from Ornithine in Animals
Ornithine comes from the urea cycle. In bacteria, ornithine has a special synthesis pathway. FIGURE 22–12 Biosynthesis of proline and arginine from glutamate in bacteria. All five carbon atoms of proline arise from glutamate. In many organisms, glutamate dehydrogenase is unusual in that it uses either NADH or NADPH as a cofactor. The same may be true of other enzymes in these pathways. The γ-semialdehyde in the proline pathway undergoes a rapid, reversible cyclization to ∆1-pyrroline-5-carboxylate (P5C), with the equilibrium favoring P5C formation. Cyclization is averted in the ornithine/arginine pathway by acetylation of the α-amino group of glutamate in the first step and removal of the acetyl group after the transamination. Although some bacteria lack arginase and thus the complete urea cycle, they can synthesize arginine from ornithine in steps that parallel the mammalian urea cycle, with citrulline and argininosuccinate as intermediates (see Fig. 18–10).

Serine Derives from 3-Phosphoglycerate of Glycolysis
Same pathway in all organisms so far Requires Glu as source of NH2 group Oxidation  transamination  dephosphorylation to yield serine FIGURE 22-14 Biosynthesis of serine from 3-phosphoglycerate and of glycine from serine in all organisms. Glycine is also made from CO2 and NH+4 by the action of glycine synthase, with N5,N10-methylenetetra-hydrofolate as methyl group donor (see text).

Glycine Derives from Serine
Carbon removed using tetrahydrofolate (H4 folate) to accept the C atom and pyridoxal phosphate (PLP) Reaction uses serine hydroxymethyltransferase FIGURE 22-14 Biosynthesis of serine from 3-phosphoglycerate and of glycine from serine in all organisms. Glycine is also made from CO2 and NH+4 by the action of glycine synthase, with N5,N10-methylenetetra-hydrofolate as methyl group donor (see text).

Cysteine Also Derives from Serine
In bacteria and plants, sulfates are the source of S. FIGURE 22–15 Biosynthesis of cysteine from serine in bacteria and plants. The origin of reduced sulfur is shown in the pathway on the right.

Biosynthesis of Cys from Homocysteine and Ser in Mammals
In mammals, sulfur is recycled from methionine degradation. FIGURE 22–16 Biosynthesis of cysteine from homocysteine and serine in mammals. The homocysteine is formed from methionine, as described in the text.

Oxaloacetate Yields Asp, Which Yields Asn, Met, Lys, and Thr

Oxaloacetate Yields Asp, Which Yields Asn, Met, Lys, and Thr
Refer to Figure Aspartate is formed from transamination of oxaloacetate. Aspartate is the root of the other amino acids.

Pyruvate Yields Ala, Val, Leu, and Ile

Aromatic Amino Acids Derive from Phosphoenolpyruvate and Erythrose 4-Phosphate
Very complicated chemistry! Rings must be synthesized and closed and then oxidized to create double bonds. Chorismate is a common intermediate.

Biosynthesis of Chorismate, a Key Intermediate in Aromatic Amino Acid Biosynthesis
FIGURE 22-18 Biosynthesis of chorismate, an intermediate in the synthesis of aromatic amino acids in bacteria and plants. All carbons are derived from either erythrose 4-phosphate (light purple) or phosphoenolpyruvate (light red). Note that the NAD+ required as a cofactor in step 2 is released unchanged; it may be transientlyreduced to NADH during the reaction, with formation of an oxidized reaction intermediate. Step 6 is competitively inhibited by glyphosate(-COO-CH2-NH-CH2-PO3 2-), the active ingredient in the widely used herbicide Roundup. The herbicide is relatively nontoxic to mammals, which lack this biosynthetic pathway. The intermediates quinate and shikimate are named after the plants in which they have been found to accumulate.

His Derives from PPP Metabolite Ribose 5-Phosphate
FIGURE 22-22 Biosynthesis of histidine in bacteria and plants. Atoms derived from PRPP and ATP are shaded light red and blue, respectively. Two of the histidine nitrogens are derived from glutamine and glutamate (green). Note that the derivative of ATP remaining after step 5 (AICAR)is an intermediate in purine biosynthesis (see Fig , step 9), soATP is rapidly regenerated.

Several Pathways Share 5-Phosphoribosyl-1-Pyrophosphate (PRPP) as an Intermediate
Synthesized from ribose 5-phosphate of PPP via ribose phosphate pyrophosphokinase a highly regulated allosteric enzyme

Regulation of Amino Acid Biosynthesis
Multilayered approach: Often, more than one mechanism of regulation is utilized. feedback inhibition of products use of isozymes for regulation of specific pathways

Feedback Inhibition in Ile Synthesis from Thr
FIGURE 22–23 Allosteric regulation of isoleucine biosynthesis. The first reaction in the pathway from threonine to isoleucine is inhibited by the end product, isoleucine. This was one of the first examples of allosteric feedback inhibition to be discovered. The steps from α-ketobutyrate to isoleucine correspond to steps 18 through 21 in Figure 22–17 (five steps, because 19 is a two-step reaction).

Use of Isozymes Is Another Important Means of Regulation
Example: Asp can lead to Lys, Met, Thr, and Ile. Use of isozymes, all regulated by different effectors, allows E. coli to produce the amino acids when needed. Example: At step 1, isozyme A1 is inhibited if Ile is high, but not if Met or Thr are high. Only the A1 isozyme is inhibited by Ile at this step.

Regulation of Aspartate-Derived Pathways
FIGURE 22–24 Interlocking regulatory mechanisms in the biosynthesis of several amino acids derived from aspartate in E. coli. Three enzymes (A, B, C) have either two or three isozyme forms, indicated by numerical subscripts. In each case, one isozyme (A2, B1, and C2) has no allosteric regulation; these isozymes are regulated by changes in the amount of enzyme synthesized (Chapter 28). Synthesis of isozymes A2 and B1 is repressed when methionine levels are high, and synthesis of isozyme C2 is repressed when isoleucine levels are high. Enzyme A is aspartokinase; B, homoserine dehydrogenase; C, threonine dehydratase.

Important Metabolites Are Derived From Amino Acids
Porphyrin rings (e.g., heme) Phosphocreatine Glutathione Neurotransmitters and signaling molecules Cell-wall constituents FIGURE 22–24 Interlocking regulatory mechanisms in the biosynthesis of several amino acids derived from aspartate in E. coli. Three enzymes (A, B, C) have either two or three isozyme forms, indicated by numerical subscripts. In each case, one isozyme (A2, B1, and C2) has no allosteric regulation; these isozymes are regulated by changes in the amount of enzyme synthesized (Chapter 28). Synthesis of isozymes A2 and B1 is repressed when methionine levels are high, and synthesis of isozyme C2 is repressed when isoleucine levels are high. Enzyme A is aspartokinase; B, homoserine dehydrogenase; C, threonine dehydratase.

Glycine or Glutamate Is the Precursor to Porphyrins
Porphyrin makes up the heme of hemoglobin, cytochromes, myoglobin. In higher animals, porphyrin arises from reaction of glycine with succinyl-CoA. In plants and bacteria, glutamate is the precursor. The pathway generates two molecules of the important intermediate -aminolevulinate. Porphobilinogen is another important intermediate.

Synthesis of -Aminolevulinate in Higher Eukaryotes
FIGURE 22–25a Biosynthesis of δ-aminolevulinate. (a) In most animals, including mammals, δ-aminolevulinate is synthesized from glycine and succinyl-CoA. The atoms furnished by glycine are shown in red.

Synthesis of -Aminolevulinate in Plants and Bacteria
FIGURE 22–25b Biosynthesis of δ-aminolevulinate. (b) In bacteria and plants, the precursor of δ-aminolevulinate is glutamate.

Synthesis of Heme from -Aminolevulinate
Two molecules of -aminolevulinate condense to form porphobilinogen. Four molecules of porphobilinogen combine to form protoporphyrin. Fe ion is inserted into protoporphyrin with the enzyme ferrochelatase. FIGURE 22–26 Biosynthesis of heme from δ-aminolevulinate. Ac represents acetyl (—CH2COO–); Pr, propionyl (—CH2CH2COO–).

Defects in Heme Biosynthesis
Most animals synthesize their own heme. Mutations or misregulaton of enzymes in the heme biosynthesis pathway lead to porphyrias. Precursors accumulate in red blood cells, body fluids, and liver. Accumulation of precursor uroporphyrinogen I Urine becomes discolored (pink to dark purplish depending on light, heat exposure). Teeth may show red fluorescence under UV light. Skin is sensitive to UV light. There is a craving for heme. Explored as possible biochemical basis for vampire myths

Heme Is the Source of Bile Pigments
Heme from degradation of erythrocytes is degraded to bilirubin in two steps: Heme oxygenase linearizes heme to create biliverdin, a green compound (seen in a bruise). Biliverdin reductase converts biliverdin to bilirubin, a yellow compound that travels bound to serum albumin in the bloodstream. major pigment of urine (degradation to urobilin) further degraded by intestinal microbiota to stercobilin

Formation and Breakdown of Bilirubin
FIGURE 22–27 Bilirubin and its breakdown products. M represents methyl; V, vinyl; Pr, propionyl; E, ethyl. For ease of comparison, these structures are shown in linear form, rather than in their correct stereochemical conformations.

Jaundice Is Caused by Bilirubin Accumulation
Jaundice (yellowish pigmentation of skin, whites of eyes, etc.) can result from: impaired liver (in liver cancer, hepatitis) blocked bile secretion (due to gallstones, pancreatic cancer) insufficient glucouronyl bilirubin transferase to process bilirubin (occurs in infants) treated with UV to cause photochemical breakdown of bilirubin

Gly and Arg Are Precursors of Creatine and Phosphocreatine
Phosphocreatine is hydrolyzed for energy in muscle. Gly and Arg combine, then S-adenosyl-methionine (Ado-Met) acts as a methyl donor. FIGURE 1315 Hydrolysis of phosphocreatine. Breakage of the PON bond in phosphocreatine produces creatine, which is stabilized by formation of a resonance hybrid. The other product, Pi, is also resonance stabilized.

Biosynthesis of Creatine and Phosphocreatine
Requires glycine, arginine, and S-adenosyl-methionine Phosphocreatine can be phosphorylated by ATP for use as a stored energy source. FIGURE 22–28 Biosynthesis of creatine and phosphocreatine. Creatine is made from three amino acids: glycine, arginine, and methionine. This pathway shows the versatility of amino acids as precursors of other nitrogenous biomolecules.

Glutathione (GSH) Derives from Glu, Cys, and Gly
GSH is present in most cells at high amounts. Reducing agent/antioxidant keeps proteins, metal cations reduced keeps redox enzymes in reduced state removes toxic peroxides Oxidized to a dimer using disulfide bond (GSSG) FIGURE 2229 Glutathione metabolism. (a) Biosynthesis of glutathione. (b) Oxidized form of glutathione.

d-Amino Acids in Bacteria Arise from Racemases
Bacterial peptidoglycans contain d-Al and d-Glu. Racemases act on d-amino acids and use PLP as a cofactor. Racemase inhibitors are used/studied as antibiotic targets. cycloserine for tuberculosis l-fluoroalanine as a general antibiotic

Aromatic Amino Acids Are Precursors to Plant Lignins, Hormones, and Natural Products
Lignin (rigid polymer in plants) from Phe and Tyr Auxin (growth hormone indole-3-acetate) from Trp Other natural products: spices (nutmeg, vanilla), alkaloids (morphine), and so on

Biosynthesis of Auxin from Trp and Cinnamate from Phe
FIGURE 22–30 Biosynthesis of two plant substances from amino acids. (a) Indole-3-acetate (auxin) and (b) cinnamate (cinnamon flavor).

Some Neurotransmitters Are Derived from Amino Acids
FIGURE 22–31 Biosynthesis of some neurotransmitters from amino acids. The key step is the same in each case: a PLP-dependent decarboxylation (shaded light red). All are decarboxylated using PLP dependent enzymes.

Arg Is the Precursor for Nitric Oxide (NO)
Mid-80’s discovery that pollutant NO played important role in blood pressure regulation, blood clotting, and so on Synthesized from Arg via nitric oxide synthase using NADPH enzyme similar to cyt P450 reductase stimulated by interaction with Ca2+ and calmodulin FIGURE 22-33 Biosynthesis of nitric oxide. Both steps are catalyzed by nitric oxide synthase. The nitrogen of the NO is derived from the guanidinium group of arginine.

Nucleotide Biosynthesis
Nucleotides can be synthesized de novo (“from the beginning”) from amino acids, ribose-5-phosphate, CO2, and NH3. Nucleotides can be salvaged from RNA, DNA, and cofactor degradation. Many parasites (e.g., malaria) lack de novo biosynthesis pathways and rely exclusively on salvage. Compounds that inhibit salvage pathways are promising antiparasite drugs.

De Novo Biosynthesis of Nucleotides
Approximately the same in all organisms studied Bases synthesized while attached to ribose Glu provides most amino groups. Gly is precursor for purines Asp is precursor for pyrimidines Nucleotide pools are kept low, so cells must continually synthesize them. This synthesis may actually limit rates of transcription and replication.

Origin of Ring Atoms in Purines
FIGURE 22–34 Origin of the ring atoms of purines. This information was obtained from isotopic experiments with 14C- or 15N-labeled precursors. Formate is supplied in the form of N10-formyltetrahydrofolate.

De Novo Biosynthesis of Purines Begins with PRPP
Adenine and guanine are synthesized as AMP and GMP. Synthesis begins with reaction of 5-phosphoribosyl 1-pyrophosphate (PRPP) with Glu. Purine ring builds up following the addition of three carbons from glycine. The first intermediate with a full purine ring is inosinate (IMP).

Construction of IMP FIGURE 22–35 (part 1) De novo synthesis of purine nucleotides: construction of the purine ring of inosinate (IMP). Each addition to the purine ring is shaded to match Figure 22–34. After step 2, R symbolizes the 5-phospho-D-ribosyl group on which the purine ring is built. Formation of 5-phosphoribosylamine (step 1) is the first committed step in purine synthesis. Note that the product of step 9, AICAR, is the remnant of ATP released during histidine biosynthesis (see Fig. 22–22, step 5). Abbreviations are given for most intermediates to simplify the naming of the enzymes. Step 6a is the alternative path from AIR to CAIR occurring in higher eukaryotes.

FIGURE 22–35 (part 2) De novo synthesis of purine nucleotides: construction of the purine ring of inosinate (IMP). Each addition to the purine ring is shaded to match Figure 22–34. After step 2, R symbolizes the 5-phospho-D-ribosyl group on which the purine ring is built. Formation of 5-phosphoribosylamine (step 1) is the first committed step in purine synthesis. Note that the product of step 9, AICAR, is the remnant of ATP released during histidine biosynthesis (see Fig. 22–22, step 5). Abbreviations are given for most intermediates to simplify the naming of the enzymes. Step 6a is the alternative path from AIR to CAIR occurring in higher eukaryotes.

Synthesis of AMP and GMP from IMP
Note that ATP is used to phosphorylate GMP precursor, while GTP is used to phosphorylate AMP precursor. FIGURE 22–36 Biosynthesis of AMP and GMP from IMP.

Regulation of Purine Biosynthesis in E
Regulation of Purine Biosynthesis in E. coli Largely Consists of Feedback Inhibition Four Major Mechanisms Glutamine-PRPP amidotransferase is inhibited by end-products IMP, AMP, and GMP. Excess GMP inhibits formation of xanthylate from inosinate by IMP dehydrogenase. GMP and AMP concentrations inhibit phosphorylation steps. PRPP synthesis is inhibited by ADP and GDP. FIGURE 22-37 Regulatory mechanisms in the biosynthesis of adenine and guanine nucleotides in E. coli. Regulation of these pathways differs in other organisms.

De Novo Synthesis of Pyrimidine Nucleotides (1)
Unlike purine synthesis, pyrimidine synthesis proceeds by first making the pyrimidine ring (in the form of orotate) and then attaching it to ribose 5-phosphate. Aspartate and carbamoyl phosphate provide the atoms for the ring structure. FIGURE 22–38 De novo synthesis of pyrimidine nucleotides: biosynthesis of UTP and CTP via orotidylate. The pyrimidine is constructed from carbamoyl phosphate and aspartate. The ribose 5-phosphate is then added to the completed pyrimidine ring by orotate phosphoribosyltransferase. The first step in this pathway (not shown here; see Fig. 18–11a) is the synthesis of carbamoyl phosphate from CO2 and NH+4. In eukaryotes, the first step is catalyzed by carbamoyl phosphate synthetase II.

After addition of ribose-5-phosphate via PRPP, the resulting nucleotide (orotidylate) is decarboxylated to form uridylate (UMP), the first possible pyrimidine. FIGURE 22–38 De novo synthesis of pyrimidine nucleotides: biosynthesis of UTP and CTP via orotidylate. The pyrimidine is constructed from carbamoyl phosphate and aspartate. The ribose 5-phosphate is then added to the completed pyrimidine ring by orotate phosphoribosyltransferase. The first step in this pathway (not shown here; see Fig. 18–11a) is the synthesis of carbamoyl phosphate from CO2 and NH+4. In eukaryotes, the first step is catalyzed by carbamoyl phosphate synthetase II.

UMP is phosphorylated to UTP. After formation of UTP, amination can convert UTP to CTP. FIGURE 22–38 De novo synthesis of pyrimidine nucleotides: biosynthesis of UTP and CTP via orotidylate. The pyrimidine is constructed from carbamoyl phosphate and aspartate. The ribose 5-phosphate is then added to the completed pyrimidine ring by orotate phosphoribosyltransferase. The first step in this pathway (not shown here; see Fig. 18–11a) is the synthesis of carbamoyl phosphate from CO2 and NH+4. In eukaryotes, the first step is catalyzed by carbamoyl phosphate synthetase II.

Regulation of Pyrimidine Biosynthesis Is Also via Feedback Inhibition
ATCase is inhibited by end-product CTP and is accelerated by ATP. FIGURE 22–40 Allosteric regulation of aspartate transcarbamoylase by CTP and ATP. Addition of 0.8 mM CTP, the allosteric inhibitor of ATCase, increases the K0.5 for aspartate (lower curve) thereby reducing the rate of conversion of aspartate to N-carbamoylaspartate. ATP at 0.6 mM fully reverses this inhibition by CTP (middle curve).

Ribonucleotides Are Precursors to Deoxyribonucleotides
2’C-OH bond is directly reduced to 2’-H bond… without activating the carbon! catalyzed by ribonucleotide reductase Mechanism: Two H atoms are donated by NADPH and carried by proteins thioredoxin or glutaredoxin.

Reduction of Ribonucleotides by Ribonucleotide Reductase
NADPH serves as the electron donor. Funneled through glutathione (a) or thioredoxin pathways (b) FIGURE 22–41 Reduction of ribonucleotides to deoxyribonucleotides by ribonucleotide reductase. Electrons are transmitted (blue arrows) to the enzyme from NADPH via (a) glutaredoxin or (b) thioredoxin. The sulfide groups in glutaredoxin reductase are contributed by two molecules of bound glutathione (GSH; GSSG indicates oxidized glutathione). Note that thioredoxin reductase is a flavoenzyme, with FAD as prosthetic group.

Structure of Ribonucleotide Reductase
FIGURE 22–42 Ribonucleotide reductase. (a) A schematic diagram of the subunit structures. The catalytic subunit (α; also called R1) contains the two regulatory sites described in Fig. 22–44 and two Cys residues central to the reaction mechanism. The radical-generation subunits β (also called R2) contain the critical Tyr122 residue and the binuclear iron center. (b) Structures of α2 and β2, give the likely structure of α2β2. (derived from PDB ID 3UUS). (c) The likely path of radical formation from the initial Tyr122 in a β subunit to the active-site Cys439, which is used in the mechanism shown in Figure 22–43. Several aromatic amino acid residues participate in long-range radical transfer from the point of its formation at Tyr122 to the active site, where the nucleotide substrate is bound.

Proposed Ribonucleotide Reductase Mechanism Involves Free Radicals
Most forms of enzyme have two catalytic/regulatory subunits and two radical-generating subunits. contain Fe3+ and dithiol groups enzyme creates stable Tyr radical to abstract H from sugar A 3’-ribonucleotide radical forms. 2’-OH is protonated to help eliminate H2O and form a radical-stabilized carbocation. Electrons are transferred to the 2’-C.

Proposed Mechanism for Ribonucleotide Reductase
MECHANISM FIGURE 22–43 Proposed mechanism for ribonucleotide reductase. In the enzyme of E. coli and most eukaryotes, the active thiol groups are on the α subunit; the active-site radical (—X•) is on the β subunit and in E. coli is probably a thiyl radical of Cys439 (see Fig. 22–42).

Ribonucleotide Reductase Has Two Types of Regulatory Sites
One type affects activity. ATP activates, dATP inhibits The other type affects substrate specificity in order to maintain balanced pools of nucleotides. If ATP or dATP high  less specificity for adenine and MORE specificity for UDP and CDP, and so on. Enzyme oligomerizes to accomplish this change.

Regulation of Ribonucleotide Reductase by dNTPs
FIGURE 22–44 Regulation of ribonucleotide reductase by deoxynucleoside triphosphates. The overall activity of the enzyme is affected by binding at the primary regulatory site (left). The substrate specificity of the enzyme is affected by the nature of the effector molecule bound at the second type of regulatory site, the substrate-specificity site (right). The diagram indicates inhibition or stimulation of enzyme activity with the four different substrates. The pathway from dUDP to dTTP is described later (see Figs. 22–46, 22–47).

dTMP Is Made from dUTP Roundabout pathway… dUTP is made.
dUTP  to dUMP by dUTPase dUMP  dTMP by thymidylate synthase - adds a methyl group from tetrahydrofolate Thymidylate synthase is a target for some anticancer drugs.

Biosynthesis of dTMP FIGURE 22–46 Biosynthesis of thymidylate (dTMP). The pathways are shown beginning with the reaction catalyzed by ribonucleotide reductase. Figure 22–47 gives details of the thymidylate synthase reaction.

Conversion of dUMP to dTMP by Thymidylate Synthase
FIGURE 22–47 Conversion of dUMP to dTMP by thymidylate synthase and dihydrofolate reductase. Serine hydroxymethyltransferase is required for regeneration of the N5,N10-methylene form of tetrahydrofolate. In the synthesis of dTMP, all three hydrogens of the added methyl group are derived from N5,N10-methylenetetrahydrofolate (light red and gray).

Folic Acid Deficiency Leads to Reduced Thymidylate Synthesis
Folic acid deficiency is widespread, especially in nutritionally poor populations. Reduced thymidylate synthesis causes uracil to be incorporated into DNA. Repair mechanisms remove the uracil by creating strand breaks that affect the structure and function of DNA. associated with cancer, heart disease, neurological impairment

Catabolism of Purines Dephosphorylation (via 5’-nucleotidase)
Deamination and hydrolysis of ribose lead to production of xanthine. Hypoxanthine and xanthine are then oxidized into uric acid by xanthine oxidase. Spiders and other arachnids lack xanthine oxidase. FIGURE 22–48 Catabolism of purine nucleotides. Note that primates excrete much more nitrogen as urea via the urea cycle (Chapter 18) than as uric acid from purine degradation. Similarly, fish excrete much more nitrogen as NH+4 than as urea produced by the pathway shown here.

Conversion of Uric Acid to Allantoin, Allantoate, and Urea
Degree of further oxidation of uric acid is organism dependent. FIGURE 22–48 Catabolism of purine nucleotides. Note that primates excrete much more nitrogen as urea via the urea cycle (Chapter 18) than as uric acid from purine degradation. Similarly, fish excrete much more nitrogen as NH+4 than as urea produced by the pathway shown here.

Excess Uric Acid Seen in Gout
Painful joints (often in toes) due to deposits of sodium urate crystals Primarily affects males May involve genetic under-excretion of urate and/or may involve overconsumption of fructose Treated with avoidance of purine-rich foods (seafood, liver) or avoidance of fructose Also treated with xanthine oxidase inhibitor allopurinol

Allopurinol Inhibits Xanthine Oxidase
FIGURE 22–50 Allopurinol, an inhibitor of xanthine oxidase. Hypoxanthine is the normal substrate of xanthine oxidase. Only a slight alteration in the structure of hypoxanthine (shaded light red) yields the medically effective enzyme inhibitor allopurinol. At the active site, allopurinol is converted to oxypurinol, a strong competitive inhibitor that remains tightly bound to the reduced form of the enzyme.

Catabolism of Pyrimidines
Leads to NH4+ and urea Can produce intermediates of CAC Example: Thymine is degraded to succinyl-CoA.

Purine and Pyrimidine Bases Are Recycled by Salvage Pathways
Free bases, released in metabolism, are reused. Example: Adenine reacts with PRPP to form the adenine nucleotide AMP. catalyzed by adenosine phosphoribosyltransferase The brain is especially dependent on salvage pathways. The lack of hypoxanthine-guanine phosphoribosyltransferase leads to Lesch-Nyhan syndrome with neurological impairment and finger-and-toe-biting behavior.

Many Chemotherapeutic Agents Target Nucleotide Biosynthesis
Glutamine analogs: azaserine, acivicin inhibit glutamine amidotransferases Fluorouracil converted by salvage pathway into FdUMP, which inhibits thymidylate synthase Methotrexate and aminopterin inhibit dihydrofolate reductase (competitive inhibitors)

Antibiotics Also Target Nucleotide Biosynthesis
Allopurinol, and so on studied against African sleeping sickness (trypanosomiasis) because the trypanosomes lack enzymes for de novo nucleotide synthesis Trimethoprim inhibits bacterial dihydrofolate reductase but binds human enzyme several orders of magnitude less strongly

Chapter Summary In this chapter, we learned:
methods for activation of molecular nitrogen to nitrates, nitrates, and ammonia glutamine serves as the primary ammonia donor in amino acid biosynthesis the 20 common amino acids are synthesized from -ketoglutarate, 3-phosphoglycerate, oxaloacetate, pyruvate, phosphoenolpyruvate, erythrose 4-phosphate, and ribose-5-phosphate nucleotides can be synthesized either de novo from simple precursors, or reassembled from scavenged nucleobases purines are synthesized on the ribose, while pyrimidine rings are assembled prior to attachment to ribose ribonucleotides are constructed prior to modification to product deoxyribonucleotides the purine degradation pathway in most organisms leads to uric acid, but the fate of uric acid is species-specific

22| Biosynthesis of Amino Acids, Nucleotides, and Related Molecules

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22| Biosynthesis of Amino Acids, Nucleotides, and Related Molecules

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Presentation on theme: "22| Biosynthesis of Amino Acids, Nucleotides, and Related Molecules"— Presentation transcript:

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