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Elevated homocysteine levels have been linked to cardiovascular and neurologic disease. Homocysteine

levels can accumulate in a number of ways, which are related to both folic acid and vitamin B12

metabolism. Homocysteine is derived from SAH, which arises when SAM donates a methyl group (Fig.

38.10). Because SAM is frequently donating methyl groups, there is constant production of SAH, which

leads to constant production of homocysteine. Recall from Chapter 37 that homocysteine has two

biochemical fates. The homocysteine produced can be either remethylated to methionine or condensed

with serine to form cystathionine. There are two routes to methionine production. The major one is

methylation by N5-methyl-FH4, which requires vitamin B12. The liver also contains a second pathway in

which betaine (derived from choline) can donate a methyl group to homocysteine to form methionine, but

this is a minor pathway (see Section II of this chapter). The conversion of homocysteine to cystathionine

requires pyridoxal phosphate (PLP). Thus, if an individual is deficient in vitamin B12, the conversion of

homocysteine to methionine by the major route is inhibited. This directs homocysteine to produce

cystathionine, which eventually produces cysteine. As cysteine levels accumulate, the enzyme that makes

cystathionine undergoes feedback inhibition, and that pathway is also inhibited (see Fig. 38.10). This,

overall, leads to accumulation of homocysteine, which is released into the blood. Many health food stores now sell SAMe, a stabilized version of SAM. SAMe has been hypothesized to relieve depression because the synthesis of certain neurotransmittersrequires methylation by SAM (see Chapter 46). This has led to the hypothesis that by increasing

SAM levels in the nervous system, the biosynthesis of these neurotransmitters will be accelerated.

This in turn might alleviate the feelings of depression. There have been reports in the literature

indicating that this may occur, but its efficacy as an antidepressant must be confirmed. The major

questions that must be addressed include the stability of SAMe in the digestive system and the

level of uptake of SAMe by cells of the nervous system.

Homocysteine also accumulates in the blood if a mutation is present in the enzyme that converts

N5,N10-methylene-FH4 to N5-methyl-FH4. When this occurs, the levels of N5-methyl-FH4 are too low to

allow homocysteine to be converted to methionine. The loss of this pathway, coupled with the feedback

inhibition by cysteine on cystathionine formation, also leads to elevated homocysteine levels in the blood.

A third way in which serum homocysteine levels can be elevated is by a mutated cystathionine β-

synthase or a deficiency in vitamin B6, the required cofactor for that enzyme. These defects block the

ability of homocysteine to be converted to cystathionine, and the homocysteine that does accumulate

cannot all be accommodated by conversion to methionine. Thus, an accumulation of homocysteine results.

C. Neural Tube Defects

Folate deficiency during pregnancy has been associated with an increased risk of neural tube defects in

the developing fetus. This risk is significantly reduced if women take folic acid supplements

periconceptually. The link between folate deficiency and neural tube defects was first observed in women

with hyperhomocysteinemia brought about by a thermolabile variant of N5,N10-methylene-FH4 reductase.

This form of the enzyme, which results from a single nucleotide change (C to T) in position 677 of the

gene that encodes the protein, is less active at body temperature than at lower temperatures. This results

in a reduced level of N5-methyl-FH4 being generated and, therefore, an increase in the levels of

homocysteine. Along with the elevated homocysteine, the women were also folate-deficient. The folate

deficiency and the subsequent inhibition of DNA synthesis leads to neural tube defects. The elevated

homocysteine is one indication that such a deficit is present. These findings have led to the

recommendation that women considering getting pregnant begin taking folate supplements before

conception occurs and during pregnancy. The U.S. Department of Agriculture has, in fact, mandated that

folate be added to flour-containing products in the United States. D. Folate Deficiencies and DNA Synthesis

Folate deficiencies result in decreased availability of the deoxythymidine and purine nucleotides that

serve as precursors for DNA synthesis. The decreased concentrations of these precursors affect not only

the DNA synthesis that occurs during replication before cell division but also the DNA synthesis that

occurs as a step in the processes that repair damaged DNA. Decreased methylation of dUMP to form dTMP, a reaction that requires N5,N10-methylene-FH4 as a

coenzyme (see Fig. 38.5), leads to an increase in the intracellular dUTP/deoxythymidine triphosphate

(dTTP) ratio. This ratio change causes a significant increase in the incorporation of uracil into DNA.Although much of this uracil can be removed by DNA-repair enzymes, the lack of available dTTP blocks

the step of DNA repair that is catalyzed by DNA polymerase. The result is fragmentation of DNA as well

as blockade of normal DNA replication.

These abnormal nuclear processes are responsible for the clumping and polysegmentation seen in the

nuclei of neutrophilic leukocytes in the bone marrow and in the peripheral blood of patients with

megaloblastic anemia caused by a primary folate deficiency or one that is secondary to B12 deficiency.

The abnormalities in DNA synthesis and repair lead to an irreversible loss of the capacity for cell

division and eventually to cell death. V. Choline and One-Carbon Metabolism

Other compounds involved in one-carbon metabolism are derived from degradation products of choline.

Choline, an essential component of certain phospholipids, is oxidized to form betaine aldehyde, which is

further oxidized to betaine (trimethylglycine). In the liver, betaine can donate a methyl group to

homocysteine to form methionine and dimethylglycine. This allows the liver to have two routes for

homocysteine conversion to methionine. This is in contrast to the nervous system, which only expresses

the primary B12-requiring pathway. Under conditions in which SAM accumulates, glycine can be

methylated to form sarcosine (N-methylglycine). This route of glycine metabolism is used when

methionine levels are high and excess methionine needs to be metabolized (Fig. 38.11).

CLINICAL COM M ENTS

Jean T. Jean T. developed a folate deficiency and is on the verge of developing a

cobalamin

(vitamin B12) deficiency as a consequence of prolonged, moderately severe malnutrition related to

chronic alcoholism. Before folate therapy is started, the physician must ascertain that the megaloblastic

anemia is not caused by a pure B12 deficiency or a combined deficiency of folate and B12.

If folate is given without cobalamin to a B12-deficient patient, the drug only partially corrects the

megaloblastic anemia because it will “bypass” the methyl-folate trap and provide adequate FH4coenzyme for the conversion of dUMP to dTMP and for a resurgence of purine synthesis. As a result,

normal DNA synthesis, DNA repair, and cell division occur. However, the neurologic syndrome, resulting

from hypomethylation in nervous tissue, and accumulation of methylmalonic acid, may progress unless the

physician realizes that B12 supplementation is required. In Jean’s case, in which the serum B12

concentration was borderline low and in which the dietary history supported the possibility of a B12

deficiency, a combination of folate and B12 supplements is required to avoid this potential therapeutic

trap.

Clark T. Clark T. continued to do well and returned faithfully for his regular colonoscopic

examinations.

Beatrice T. Beatrice T. was diagnosed with a decreased ability to absorb dietary B12. One of the

consequences of aging is a reduced acid production by the gastric mucosa (atrophic gastritis),

which limits the ability of pepsin to work on dietary protein. Reduced pepsin efficiency then reduces the

amount of bound B12 released from dietary protein, as a result of which the B12 is not available for

absorption. Her condition can be treated by taking high-dose vitamin B12 supplements orally.

BIOCHEM ICAL COM M ENTS

A Potential Mechanism Relating to Folate Deficiencies and Neural Tube Defects. As indicated

in Section IV.C of this chapter, neural tube defects in newborns have been associated with folate

deficiency during pregnancy. Although the mechanism leading to neural tube defects is vague, new

research has indicated that the induction of micro RNAs (miRNAs) may play a role in altering the normal

developmental pattern of neural tube closure.

Under conditions of a folate deficiency, hypomethylation occurs in the nervous system, affecting

membrane phospholipid biosynthesis (such as phosphatidylcholine), myelin basic protein (see Chapter

46), and neurotransmitter biosynthesis (see Chapter 46). The reduced levels of neurotransmitters may

interfere with normal gene expression during embryogenesis. DNA methylation is also reduced, owing to

reduced SAM levels when folate is limiting. Hypomethylation is also the result of increased levels of

SAH, which accumulates during a folate deficiency. SAH will inhibit DNA methyltransferase enzymes by

tightly binding to the enzyme, and preventing the normal substrate, SAM, from binding to the enzyme. The

enzymes affinity for SAH is higher than that of SAM, contributing to the hypomethylation observed.

miRNA genes are frequently in close proximity to CpG islands in DNA, and it has been predicted that

alterations in cytosine methylation may be a means of regulating miRNA expression.

Experimentally, a

cell line in which two DNA methyltransferase genes were knocked out (inactivated) resulted in a

significant reduction in global genomic methylation and the differential expression of 13 miRNAs (seven

of those genes were overexpressed, whereas the other six displayed a reduction in expression). A similar

result was obtained with another cell line that was placed in folate-deficient media; global

hypomethylation and alterations in miRNA expression were observed.

As an example, miR-222 was identified as a potential miRNA that is upregulated under conditions of

folate deprivation. A predicted target of miR-222 is the DNMT-1 gene (a DNA methyltransferase), whose

activity is critical for the maintenance of methylation patterns in DNA. Overexpression of miR-222 wouldreduce DNMT-1 gene expression, thereby altering methylation patterns in the cell. A reduction of DNMT-

1 activity has been shown to increase the expression of a number of genes, including β-catenin (see

Chapter 18). This will lead to enhanced cell proliferation and inhibition of differentiation in the nervous

system (all leading to a failure to close the neural tube).

Studies such as those described previously are in their infancy but produce a promising start for

unraveling the effects of DNA methylation, and miRNA expression, on cell growth and differentiation in

the nervous system. KEY CONCEPTS

One-carbon groups at lower oxidation states than carbon dioxide (which is carried by biotin) are

transferred by reactions that involve tetrahydrofolate (FH4), vitamin B12, and S-adenosylmethionine

(SAM).

FH4 is produced from the vitamin folate and obtains one-carbon units from serine, glycine, histidine,

formaldehyde, and formic acid.

The carbon attached to FH4 can be oxidized or reduced, thus producing several different forms of

FH4. However, once a carbon has been reduced to the methyl level, it cannot be reoxidized.

The carbons attached to FH4 are known collectively as the one-carbon pool.

The carbons carried by folate are used in a limited number of biochemical reactions, but they are

very important in forming deoxythymidine monophosphate (dTMP) and the purine rings. Vitamin B12 participates in two reactions in the body: conversion of L-methylmalonyl-CoA to

succinyl-CoA and conversion of homocysteine to methionine.

SAM, formed from adenosine triphosphate (ATP) and methionine, transfers the methyl group to

precursors of a variety of methylated compounds.

Both vitamin B12 and methyl-FH4 are required in methionine metabolism; a deficiency of vitamin

B12 leads to overproduction and trapping of folate in the methyl form, leading to a functional folate

deficiency. Such deficiencies can lead to Megaloblastic anemia

Neural tube defects in newborns

Diseases discussed in this chapter are summarized in Table 38.2.REVIEW QUESTIONS—CHAPTER 38

1. Which one of the following reactions requires N5,N10-methylene-FH4 as a carbon donor?

A.Homocysteine to methionine

B.Serine to glycine

C.Betaine to dimethylglycine

D.dUMP to dTMP

E. The de novo biosynthesis of the purine ring

2.Propionic acid accumulation from amino acid degradation will result from a deficiency of which one

of the following vitamins? A. Vitamin B6

B. Biotin

C. Folic acid D. Vitamin B1 E. Vitamin B2

3.A child with an acute otitis media (middle ear infection) is treated with a sulfa antibiotic. This

medication interferes with the bacterial synthesis of which one of the following? A. Vitamin B12

B. SAM

C. Folic acid D. Vitamin B6 E. Homocysteine

4.Which one of the following forms of FH4 is required for the synthesis of methionine from

homocysteine?

A. N5,N10-Methylene-FH4

B. N5-Methyl-FH4C. N5,N10-Methenyl-FH4 D. N10-Formyl-FH4

E. N5-Formimino-FH4

5.An alternative method to methylate homocysteine to form methionine is which one of the following?

A. Using glycine and FH4 as the methyl donor B. Using dimethylglycine as the methyl donor C. Using choline as the methyl donor

D. Using sarcosine as the methyl donor E. Using betaine as the methyl donor

6.Both folic acid and B12 deficiencies will lead to the observation of hypomethylation in the nervous

system. These two cofactors are most closely linked via which one of the following proteins?

A. Transcobalamin II B. Methionine synthase

C. Cystathionine β-synthase

D. N5,N10-Methylene-FH4 reductase E. DNA methyltransferase

7.A very strict vegan, who has not eaten animal products for over 5 years or taken exogenous vitamins,

slowly develops tiredness and lethargy and also notes occasional tingling in the feet. An analysis of

total folate indicated normal amounts. In which form would this folate most likely be found?

A. N5-Methyl-FH4

B. N5,N10-Methylene-FH4 C. N10-Formyl-FH4

D. N5-Formimino-FH4 E. N5,N10-Methenyl-FH4

8.A patient presents to a clinic with symptoms of lethargy and tingling in the extremities. Bloodwork

demonstrates a megaloblastic anemia. Measurement of which one of the following metabolites in the

blood will aid in determining the cause of these symptoms in this patient? A. Lactic acid

B. Pyruvic acid C. Methionine

D. Methylmalonic acid E. Histidine

9.Which one of the following patients would be in danger of developing a vitamin B12 deficiency?

A. A lacto–ovo vegetarian

B. A patient with an inability to secrete gastrin

C.A person who had his or her duodenum surgically removed

D.A patient with Crohn’s disease affecting the terminal ileum

E.A patient with a biliary duct obstruction

10. Methotrexate is a medication that has been used as an anticancer drug and currently is used in

treatment of psoriasis and rheumatoid arthritis. This folate analog directly inhibits which one of thefollowing conversions?

A.FH2 to FH4

B.FH2 to folate

C.dUMP to dTMP

D.Serine to glycine

E.Homocysteine to methionine ANSWERS TO REVIEW QUESTIONS

1. The answer is D. The homocysteine-to-methionine reaction requires N5-methyl-FH4; serine to

glycine requires free FH4 and generates N5,N10-methylene-FH4; betaine donates a methyl group to

homocysteine to form methionine without the participation of FH4; and the purine ring requires

N10-formyl-FH4 in its biosynthesis.

2. The answer is B. Propionic acid is derived from an accumulation of propionyl-CoA. The normal

pathway for the degradation of propionyl-CoA is, first, a biotin-dependent carboxylation to Dmethylmalonyl-CoA, racemization to L-methylmalonyl-CoA, and then the B12-dependent

rearrangement to succinyl-CoA.

3. The answer is C. Sulfa drugs are analogs of para-aminobenzoic acid and prevent growth and cell

division in bacteria by interfering with the synthesis of folic acid (which is needed to produce

FH4). Humans cannot synthesize folate de novo and must obtain folate from the diet. Because of

this, the sulfa drugs do not affect human metabolism.

4. The answer is B. The only three forms of folate that transfer carbons are the N5-methyl-FH4 form,

the N5,N10-methylene-FH4 form, and the N10-formyl form. None of the other forms participates in

reactions in which the carbon is transferred. It is the N5-methyl form that transfers the methyl

group to form methionine from homocysteine.

5. The answer is E. Choline, derived from phosphatidylcholine, is converted to betaine

(trimethylglycine). Betaine can donate a methyl group to homocysteine to form methionine plus

dimethylglycine. Sarcosine is N-methylglycine, which is formed when excess SAM methylates

glycine, but it is not used as a methyl donor in this reaction.

6. The answer is B. The conversion of homocysteine to methionine, catalyzed by methionine

synthase, requires vitamin B12 as a cofactor and N5-methyl-FH4 as a substrate. This is the only

mammalian reaction in which N5-methyl-FH4 can donate its carbon. Once methionine is generated,

it is converted to SAM, which acts as a methyl donor for neurotransmitter synthesis, myelin

synthesis, phospholipid synthesis, and DNA modifications. Inability to catalyze the methionine

synthase reaction, owing to either B12 or folate deficiency, would lead to a reduction of SAM

levels and hypomethylation in the nervous system. The liver does not exhibit hypomethylation

because of the betaine pathway, in which betaine can donate a methyl group to homocysteine to

form methionine, also forming dimethylglycine once betaine loses a methyl group. Transcobalamin

II carries B12 in the circulation and does not interact with folate. Cystathionine β-synthasecatalyzes the conversion of homocysteine and serine to cystathionine and requires vitamin B6 but

not B12 or folate. N5,N10-Methylene-FH4 reductase converts N5,N10-methylene-FH4 to N5-methylFH4 and requires NADH but not vitamin B12. DNA methyltransferases require SAM to methylate

cytosine residues in DNA, but folic acid is not required as a cofactor for that reaction.

7.The answer is A. The patient is suffering from a vitamin B12 deficiency. Vitamin B12 can only be

obtained from meat and dairy products in the diet, and although the body may have a 2- to 3-year

store of the vitamin, given the patient’s diet and lack of vitamins, B12 has become deficient. The

tiredness and lethargy is owing to the development of megaloblastic anemia, and the tingling is

caused by nervous system dysfunction owing to hypomethylation and methylmalonic acid in the

nervous system. In the absence of vitamin B12, folate will be trapped as N5-methyl-FH4, because

the methionine synthase reaction will be unable to proceed. Once N5-methyl-FH4 is formed, it

cannot be converted back into the other folate forms. Because this is the most stable folate form,

the other variants of folate will slowly be converted to this form and be trapped.

8.The answer is D. The megaloblastic anemia can be caused by a deficiency of either vitamin B12

or folic acid. A B12 deficiency will block two reactions, homocysteine to methionine and

methylmalonyl-CoA to succinyl-CoA. A B12 deficiency, therefore, will lead to elevated

methylmalonic acid because the methylmalonyl-CoA produced cannot be metabolized further to

succinyl-CoA. The lack of B12 also traps folate as the N5-methyl-FH4 form (because the methyl

group cannot be transferred to homocysteine), leading to a functional folate deficiency. The folate

deficiency interferes with purine and thymidine synthesis, leading to an overall cessation of DNA

synthesis in rapidly growing cells, such as reticulocytes. This leads to megaloblast development

and ineffective erythropoiesis. If the patient had a folate deficiency, FIGLU would accumulate in

the urine (a degradation product of histidine). Accumulation of lactate, or pyruvate, does not occur

with B12 or folate deficiencies. Methionine might accumulate (because of high homocysteine

levels), but it usually stays fairly level and would not be diagnostic for determining between a B12

or a folate deficiency. Histidine also would not accumulate, as FIGLU accumulates in a folate

deficiency, and this would be used to distinguish between B12 and folate deficiencies.

9.The answer is D. Lacto–ovo vegetarians eat eggs and dairy products. Eggs are a dietary source of

vitamin B12. Stomach intrinsic factor binds released B12, but stomach gastrin has no role in B12

absorption. Vitamin B12 bound to intrinsic factor is absorbed in the terminal ileum, not the

duodenum. The ileum is involved in Crohn’s disease, which then interferes with B12 absorption.

Biliary duct obstruction would block bile acid release from the gallbladder into the intestine,

which would interfere with absorption of dietary fat but not absorption of B12, which is a watersoluble vitamin.

10. The answer is A. Methotrexate inhibits DHFR, which blocks the conversion of FH2 to FH4. The

reduction of cellular pools of FH4 would then indirectly affect reactions that require FH4. 5-FU

directly inhibits the conversion of dUMP to dTMP. The conversion of homocysteine to methionine

requires vitamin B12 and N5-methyl-FH4. The N5-methyl-FH4 levels will be low owing to the

folate being trapped as FH2 because of the direct inhibition of DHFR by methotrexate.39 Purine and Pyrimidine Metabolism

For additional ancillary materials related to this chapter, please visit thePoint. Purines and pyrimidines are required for synthesizing nucleotides and nucleic acids. These molecules

can be synthesized from scratch, de novo, or salvaged from existing bases. Dietary uptake of purine and

pyrimidine bases is low because most of the ingested nucleic acids are metabolized by the intestinal

epithelial cells.

The de novo pathway of purine synthesis is complex, consisting of 11 steps and requiring six

molecules of adenosine triphosphate (ATP) for every purine synthesized. The precursors that donate

components to produce purine nucleotides include glycine, ribose 5-phosphate, glutamine, aspartate,

carbon dioxide, and N10-formyltetrahydrofolate (Fig. 39.1). Purines are synthesized as

ribonucleotides, with the initial purine synthesized being inosine monophosphate (IMP). Adenosine

monophosphate (AMP) and guanosine monophosphate (GMP) are each derived from IMP in two-step

reaction pathways.

The purine nucleotide salvage pathway allows free purine bases to be converted into nucleotides,

nucleotides into nucleosides, and nucleosides into free bases. Enzymes included in this pathway are AMP

and adenosine deaminase (ADA), adenosine kinase, purine nucleoside phosphorylase, adenine

phosphoribosyltransferase (APRT), and hypoxanthine–guanine phosphoribosyltransferase (HGPRT). Mutations in several of these enzymes lead to serious diseases. Deficiencies in purinenucleoside phosphorylase and ADA lead to immunodeficiency disorders. A deficiency in HGPRT leads

to Lesch-Nyhan syndrome. The purine nucleotide cycle, in which aspartate carbons are converted to

fumarate to replenish tricarboxylic acid (TCA) cycle intermediates in working muscle, and the aspartate

nitrogen is released as ammonia, uses components of the purine nucleotide salvage pathway.

Pyrimidine bases are first synthesized as the free base and then converted to a nucleotide. Aspartate

and carbamoyl phosphate form all components of the pyrimidine ring. Ribose 5-phosphate, which is

converted to 5′-phosphoribosyl 1′-pyrophosphate (PRPP), is required to donate the sugar phosphate to

form a nucleotide. The first pyrimidine nucleotide produced is orotate monophosphate (OMP). The

OMP is converted to uridine monophosphate (UMP), which becomes the precursor for both cytidine

triphosphate (CTP) and deoxythymidine monophosphate (dTMP) production.

The formation of deoxyribonucleotides requires ribonucleotide reductase activity, which catalyzes

the reduction of ribose on nucleotide diphosphate substrates to 2′-deoxyribose. Substrates for the enzyme

include adenosine diphosphate (ADP), guanosine diphosphate (GDP), cytidine diphosphate (CDP), and

uridine diphosphate (UDP). Regulation of the enzyme is complex. There are two major

allosteric sites.

One controls the overall activity of the enzyme, whereas the other determines the substrate specificity of

the enzyme. All deoxyribonucleotides are synthesized using this one enzyme.

The regulation of de novo purine nucleotide biosynthesis occurs at four points in the pathway. The

enzymes PRPP synthetase, amidophosphoribosyltransferase, IMP dehydrogenase, and adenylosuccinate synthetase are regulated by allosteric modifiers because they occur at key branch

points through the pathway. Pyrimidine synthesis is regulated at the first committed step, which is the

synthesis of cytoplasmic carbamoyl phosphate, by the enzyme carbamoyl phosphate synthetase II

(CPSII).

Purines, when degraded, cannot generate energy, nor can the purine ring be substantially modified.

The end-product of purine ring degradation is uric acid, which is excreted in the urine. Uric acid has

limited solubility, and if it were to accumulate, uric acid crystals would precipitate in tissues of the body

that have a reduced temperature (such as the big toe). This condition of acute painful inflammation of

specific soft tissues and joints is called gout. Pyrimidines, when degraded, however, give rise to watersoluble compounds such as urea, carbon dioxide, and water and do not lead to a disease state if

pyrimidine catabolism is increased. THE WAITING ROOM

The initial acute inflammatory process that caused Lotta T. to experience a painful attack of gouty

arthritis responded quickly to colchicine therapy (see Chapter 10). Several weeks after the

inflammatory signs and symptoms in her right great toe subsided, Lotta was placed on allopurinol (while

continuing colchicine), a drug that reduces uric acid synthesis. Her serum uric acid level gradually fell

from a pretreatment level of 9.2 mg/dL into the normal range (2.5 to 8.0 mg/dL). She remained free of

gouty symptoms when she returned to her physician for a follow-up office visit. I. Purines and PyrimidinesAs has been seen in previous chapters, nucleotides serve numerous functions in different reaction

pathways. For example, nucleotides are the activated precursors of DNA and RNA. Nucleotides form the

structural moieties of many coenzymes (examples include nicotinamide adenine dinucleotide [NAD+],

flavin adenine dinucleotide [FAD], and coenzyme A). Nucleotides are critical elements in energy

metabolism (ATP, guanosine triphosphate [GTP]). Nucleotide derivatives are frequently activated

intermediates in many biosynthetic pathways. For example, UDP-glucose and CDP-diacylglycerol are

precursors of glycogen and phosphoglycerides, respectively. S-Adenosylmethionine carries an activated

methyl group. In addition, nucleotides act as second messengers in intracellular signaling (e.g., cyclic

adenosine monophosphate [cAMP], cyclic guanosine monophosphate [cGMP]). Finally, nucleotides and

nucleosides act as metabolic allosteric regulators. Think about all of the enzymes that have been studied

that are regulated by levels of ATP, ADP, and AMP.

Dietary uptake of purine and pyrimidine bases is minimal. The diet contains nucleic acids, and the

exocrine pancreas secretes deoxyribonuclease and ribonuclease, along with the proteolytic and lipolytic

enzymes. This enables digested nucleic acids to be converted to nucleotides. The intestinal epithelial

cells contain alkaline phosphatase activity, which converts nucleotides to nucleosides. Other enzymes

within the epithelial cells tend to metabolize the nucleosides to uric acid (which is released into the

circulation) or to salvage them for their own needs. Approximately 5% of ingested nucleotides make it

into the circulation, either as the free base or as a nucleoside. Because of the minimal dietary uptake of

these important molecules, de novo synthesis of purines and pyrimidines is required. II. Purine Biosynthesis

The purine bases are produced de novo by pathways that use amino acids as precursors and produce

nucleotides. Most de novo synthesis occurs in the liver (Fig. 39.2), and the nitrogenous bases and

nucleosides are then transported to other tissues by red blood cells. The brain also synthesizes significant

amounts of nucleotides. Because the de novo pathway requires at least six high-energy bonds per purine

produced, a salvage pathway, which is used by many cell types, can convert free bases and nucleosides to

nucleotides.A. De Novo Synthesis of the Purine Nucleotides 1. Synthesis of Inosine Monophosphate

Because purines are built on a ribose base (see Fig. 39.2), an activated form of ribose is used to initiate

the purine biosynthetic pathway. 5-Phosphoribosyl 1-pyrophosphate (PRPP) is the activated source of the

ribose moiety. It is synthesized from ATP and ribose 5-phosphate (Fig. 39.3), which is produced from

glucose through the pentose phosphate pathway (see Chapter 27). The enzyme that catalyzes this reaction,

PRPP synthetase, is a regulated enzyme (see Section II.A.5); however, this step is not the committed step

of purine biosynthesis. PRPP has many other uses, which are described as the chapter progresses.In the first committed step of the purine biosynthetic pathway, PRPP reacts with glutamine to form 5′-

phosphoribosyl 1′-amine (Fig. 39.4). This reaction, which produces nitrogen 9 of the purine ring, is

catalyzed by glutamine phosphoribosylamidotransferase, a highly regulated enzyme. In the next step of the pathway, the entire glycine molecule is added to the growing precursor. Glycine

provides carbons 4 and 5 and nitrogen 7 of the purine ring (Fig. 39.5). This step requires energy in the

form of ATP.Subsequently, carbon 8 is provided by N10-formyltetrahydrofolate (N10-formyl-FH4), nitrogen 3 by

glutamine, carbon 6 by CO2, nitrogen 1 by aspartate, and carbon 2 by N10-formyl-FH4 (see Fig. 39.1).

Note that six high-energy bonds of ATP are required (starting with ribose 5-phosphate) to synthesize the

first purine nucleotide, IMP. This nucleotide contains the base hypoxanthine joined by an N-glycosidic

bond from nitrogen 9 of the purine ring to carbon 1 of the ribose (Fig. 39.6). Hypoxanthine is not found in

DNA, but it is the precursor for the other purine bases. However, hypoxanthine is found in the anticodon

of transfer RNA molecules (see Chapter 15) and is a critical component for allowing wobble base pairs

to form.

2. Synthesis of Adenosine Monophosphate

IMP serves as the branch point from which both adenine and guanine nucleotides can be produced (see

Fig. 39.2). AMP is derived from IMP in two steps (Fig. 39.7). In the first step, aspartate is added to IMP

to form adenylosuccinate, a reaction similar to the one catalyzed by argininosuccinate synthetase in the

urea cycle. Note that this reaction requires a high-energy bond, donated by GTP.