INVESTIGATION OF BIOSYNTHESIS AND CATABOLISM OF PURINE NUCLEOTIDES. DETERMINATION OF THE END PRODUCTS OF THERE METABOLISM. INVESTIGATION OF BIOSYNTHESIS AND CATABOLISM OF PYRIMIDINE NUCLEOTIDES. DETERMINATION OF THE END PRODUCTS OF THERE METABOLISM
One of the important specialized pathways of a number of amino acids is the synthesis of purine and pyrimidine nucleotides. These nucleotides are important for a number of reasons. Most of them, not just ATP, are the sources of energy that drive most of our reactions. ATP is the most commonly used source but GTP is used in protein synthesis as well as a few other reactions. UTP is the source of energy for activating glucose and galactose. CTP is an energy source in lipid metabolism. AMP is part of the structure of some of the coenzymes like NAD and Coenzyme A. And, of course, the nucleotides are part of nucleic acids. Neither the bases nor the nucleotides are required dietary components. We can both synthesize them de novo and salvage and reuse those we already have.
There are two kinds of nitrogen-containing bases - purines and pyrimidines. Purines consist of a six-membered and a five-membered nitrogen-containing ring, fused together. Pyridmidines have only a six-membered nitrogen-containing ring. There are 4 purines and 4 pyrimidines that are of concern to us.
Adenine and guanine are found in both DNA and RNA. Hypoxanthine and xanthine are not incorporated into the nucleic acids as they are being synthesized but are important intermediates in the synthesis and degradation of the purine nucleotides.
Cytosine is found in both DNA and RNA. Uracil is found only in RNA. Thymine is normally found in DNA. Sometimes tRNA will contain some thymine as well as uracil.
If a sugar, either ribose or 2-deoxyribose, is added to a nitrogen base, the resulting compound is called a nucleoside. Carbon 1 of the sugar is attached to nitrogen 9 of a purine base or to nitrogen 1 of a pyrimidine base. The names of purine nucleosides end in -osine and the names of pyrimidine nucleosides end in -idine. The convention is to number the ring atoms of the base normally and to use l', etc. to distinguish the ring atoms of the sugar. Unless otherwise specificed, the sugar is assumed to be ribose. To indicate that the sugar is 2'-deoxyribose, a d- is placed before the name.
Adding one or more phosphates to the sugar portion of a nucleoside results in a nucleotide. Generally, the phosphate is in ester linkage to carbon 5' of the sugar. If more than one phosphate is present, they are generally in acid anhydride linkages to each other. If such is the case, no position designation in the name is required. If the phosphate is in any other position, however, the position must be designated. For example, 3'-5' cAMP indicates that a phosphate is in ester linkage to both the 3' and 5' hydroxyl groups of an adenosine molecule and forms a cyclic structure. 2'-GMP would indicate that a phosphate is in ester linkage to the 2' hydroxyl group of a guanosine. Some representative names are:
• AMP = adenosine monophosphate = adenylic acid
• CDP = cytidine diphosphate
• dGTP = deoxy guanosine triphosphate
• dTTP = deoxy thymidine triphosphate (more commonly designated TTP)
• cAMP = 3'-5' cyclic adenosine monophosphate
Nucleotides are joined together by 3'-5' phosphodiester bonds to form polynucleotides. Polymerization of ribonucleotides will produce an RNA while polymerization of deoxyribonucleotides leads to DNA.
Hydrolysis of Polynucleotides
Most, but not all, nucleic acids in the cell are associated with protein. Dietary nucleoprotein is degraded by pancreatic enzymes and tissue nucleoprotein by lysosomal enzymes. After dissociation of the protein and nucleic acid, the protein is metabolized like any other protein.
The nucleic acids are hydrolyzed randomly by nucleases to yield a mixture of polynucleotides. These are further cleaved by phosphodiesterases (exonucleases) to a mixture of the mononucleotides. The specificity of the pancreatic nucleotidases gives the 3'-nucleotides and that of the lysosomal nucleotidases gives the biologically important 5'-nucleotides.
The nucleotides are hydrolyzed by nucleotidases to give the nucleosides and Pi. This is probably the end product in the intestine with the nucleosides being the primary form absorbed. In at least some tissues, the nucleosides undergo phosphorolysis with nucleoside phosphorylases to yield the base and ribose 1-P (or deoxyribose 1-P). Since R 1-P and R 5-P are in equilibrium, the sugar phosphate can either be reincorporated into nucleotides or metabolized via the Hexose Monophosphate Pathway. The purine and pyrimidine bases released are either degraded or salvaged for reincorporation into nucleotides.
There is significant turnover of all kinds of RNA as well as the nucleotide pool. DNA doesn't turnover but portions of the molecule are excised as part of a repair process.
Purine and pyrimidine bases which are not degraded are recycled - i.e. reincorporated into nucleotides. This recycling, however, is not sufficient to meet total body requirements and so some de novo synthesis is essential. There are definite tissue differences in the ability to carry out de novo synthesis. De novo synthesis of purines is most active in liver. Non-hepatic tissues generally have limited or even no de novo synthesis. Pyrimidine synthesis occurs in a variety of tissues. For purines, especially, non-hepatic tissues rely heavily on preformed bases - those salvaged from their own intracellular turnover supplemented by bases synthesized in the liver and delivered to tissues via the blood.
"Salvage" of purines is reasonable in most cells because xanthine oxidase, the key enzyme in taking the purines all of the way to uric acid, is significantly active only in liver and intestine. The bases generated by turnover in non-hepatic tissues are not readily degraded to uric acid in those tissues and, therefore, are available for salvage. The liver probably does less salvage but is very active in de novo synthesis - not so much for itself but to help supply the peripheral tissues.
De novo synthesis of both purine and pyrimidine nucleotides occurs from readily available components.
Phosphoribosyl pyrophosphate (PRPP) is important in both, and in these pathways the structure of ribose is retained in the product nucleotide, in contrast to its fate in the tryptophan and histidine biosynthetic pathways discussed earlier. An amino acid is an important precursor in each type of pathway: glycine for purines and aspartate for pyrimidines. Glutamine again is the most important source of amino groups — in five different steps in the de novo pathways. Aspartate is also used as the source of an amino group in the purine pathways, in two steps. Two other features deserve mention. First, there is evidence, especially in the de novo purine pathway, that the enzymes are present as large, multienzyme complexes in the cell, a recurring theme in our discussion of metabolism. Second, the cellular pools of nucleotides (other than ATP) are quite small, perhaps 1% or less of the amounts required to synthesize the cell’s DNA.
Therefore, cells must continue to synthesize nucleotides during nucleic acid synthesis, and in some cases nucleotide synthesis may limit the rates of DNA replication and transcription. Because of the importance of these processes in dividing cells, agents that inhibit nucleotide synthesis have become particularly important to modern medicine. We examine here the biosynthetic pathways of purine and pyrimidine nucleotides and their regulation, the formation of the deoxynucleotides, and the degradation of purines and pyrimidines to uric acid and urea. We end with a discussion of chemotherapeutic agentsthat affect nucleotide synthesis.
De Novo Synthesis of Purine Nucleotides
The two parent purine nucleotides of nucleic acids are adenosine-monophosphate (AMP; adenylate) and guanosine-monophosphate (GMP; guanylate), containing the purine bases adenine and guanine. Figure shows the origin of the carbon and nitrogen atoms of the purine ring system, as determined by John Buchanan using isotopic tracer experiments in birds. The detailed pathway of purine biosynthesis was worked out primarily by Buchanan and G. Robert Greenberg in the 1950s.
In the first committed step of the pathway, an amino group donated by glutamine is attached at C-1 of PRPP.
The resulting 5-phosphoribosylamine is highly unstable, with a half-life of 30 seconds at pH 7.5. The purine ring is subsequently built up on this structure. The pathway described here is identical in all organisms, with the exception of one step that differs in higher eukaryotes as noted below.
The second step is the addition of three atoms from glycine. An ATP is consumed to activate the glycine carboxyl group (in the form of an acyl phosphate) for this condensation reaction:
The added glycine amino group is then formylated by N10- formyltetrahydrofolate.
A nitrogen is contributed by glutamine.
Before dehydration and ring closure yield the five-membered imidazole ring of the purine nucleus, as 5-aminoimidazole ribonucleotide.
At this point, three of the six atoms needed for the second ring in the purine structure are in place. To complete the process, a carboxyl group is first added. This carboxylation is unusual in that it does not require biotin, but instead uses the bicarbonate generally present in aqueous solutions. A rearrangement transfers the carboxylate from the exocyclic amino group to position 4 of the imidazole ring.
In higher eukaryotes, including humans, the 5-aminoimidazole ribonucleotide product is carboxylated directly to carboxyaminoimidazole ribonucleotide in one step instead of two. The enzyme catalyzing this reaction is AIR carboxylase.
Aspartate now donates its amino group in two steps formation of an amide bond, followed by elimination of the carbon skeleton of aspartate (as fumarate).
Recall that aspartate plays an analogous role in two steps of the urea cycle. The final carbon is contributed by N10-formyltetrahydrofolate and a second ring closure takes place to yield the second fused ring of the purine nucleus.
The first intermediate with a complete purine ring is inosinate (IMP).
As in the tryptophan and histidine biosynthetic pathways, the enzymes of IMP synthesis appear to be organized as large, multienzyme complexes in the cell. Once again, evidence comes from the existence of single polypeptides with several functions, some catalyzing nonsequential steps in the pathway. In eukaryotic cells ranging from yeast to fruit flies to chickens, are catalyzed by a multifunctional protein. An additional multifunctional protein catalyzes steps 10 and 11. In humans, a multifunctional enzyme combines the activities of AIR carboxylase and SAICAR synthetase.
In bacteria, these activities are found on separate proteins, but a large noncovalent complex may exist in these cells. The channeling of reaction intermediates from one enzyme to the next permitted by these complexes is probably especially important for unstable intermediates such as 5-phosphoribosylamine.
Conversion of inosinate to adenylate requires the insertion of an amino group derived from aspartate; this takes place in two reactions similar to those used to introduce N-1 of the purine ring. A crucial difference is that GTP rather than ATP is the source of the high-energy phosphate in synthesizing adenylosuccinate.
Guanylate is formed by the NAD1-requiring oxidation of inosinate at C-2, followed by addition of an amino group derived from glutamine. ATP is cleaved to AMP and PPi in the final step.
REGULATION OF PURINE NUCLEOTIDE BIOSYNTHESIS
Three major feedback mechanisms cooperate in regulating the overall rate of de novo purine nucleotide synthesis and the relative rates of formation of the two end products, adenylate and guanylate. The first mechanism is exerted on the first reaction that is unique to purine synthesis — transfer of an amino group to PRPP to form 5-phosphoribosylamine. This reaction is catalyzed by the allosteric enzyme glutamine-PRPP amidotransferase, which is inhibited by the end products IMP, AMP, and GMP. AMP and GMP act synergistically in this concerted inhibition. Thus, whenever either AMP or GMP accumulates to excess, the first step in its biosynthesis from PRPP is partially inhibited.
In the second control mechanism, exerted at a later stage, an excess of GMP in the cell inhibits formation of xanthylate from inosinate by IMP dehydrogenase, without affecting the formation of AMP. Conversely, an accumulation of adenylate inhibits formation of adenylosuccinate by adenylosuccinate synthetase, without affecting the biosynthesis of GMP. In the third mechanism, GTP is required in the conversion of IMP to AMP ( step 1 ), whereas ATP is required for conversion of IMP to GMP (step 4 ), a reciprocal arrangement that tends to balance the synthesis of the two ribonucleotides.
The final control mechanism is the inhibition of PRPP synthesis by the allosteric regulation of ribose phosphate pyrophosphokinase. This enzyme is inhibited by ADP and GDP, in addition to metabolites from other pathways of which PRPP is a starting point.
Biosynthesis of NAD+
Nicotinamide adenine dinucleotide (NAD+) and its phosphorylated analog, NADP+, are important coenzymes that participate in a number of biological processes involving electron transfer. NAD+ contains an AMP moiety as part of the molecule:
NAD+ synthesis requires nicotinate (vitamin B6), which is derived from tryptophan. In the first step, nicotinate ribonucleotide is formed from nicotinate and PRPP:
In the following steps, an AMP moiety is transferred from ATP to nicotinate ribonucleotide to form desamido-NAD+. Finally, the carboxyl group of desamido-NAD is converted to amide using glutamine as an ammonia donor:
NADP is obtained by phosphorylation of the 2'-OH of the adenine ribose by ATP in the presence of NAD+ kinase.
The end product of purine catabolism in man is uric acid. Uric acid is formed primarily in the liver and excreted by the kidney into the urine.
Nucleotides to Bases
Guanine nucleotides are hydrolyzed to the nucleoside guanosine which undergoes phosphorolysis to guanine and ribose 1-P. Man's intracellular nucleotidases are not very active toward AMP, however. Rather, AMP is deaminated by the enzyme adenylate (AMP) deaminase to IMP. In the catobilsm of purine nucleotides, IMP is further degraded by hydrolysis with nucleotidase to inosine and then phosphorolysis to hypoxanthine.
Adenosine does occur but usually arises from S-Adenosylmethionine during the course of transmethylation reactions. Adenosine is deaminated to inosine by an adenosine deaminase. Deficiencies in either adenosine deaminase or in the purine nucleoside phosphorylase lead to two different immunodeficiency diseases by mechanisms that are not clearly understood. With adenosine deaminase deficiency, both T and B-cell immunity is affected. The phosphorylase deficiency affects the T cells but B cells are normal. In September, 1990, a 4 year old girl was treated for adenosine deaminase deficiency by genetically engineering her cells to incorporate the gene. The treatment,so far, seems to be successful.
Whether or not methylated purines are catabolized depends upon the location of the methyl group. If the methyl is on an -NH2, it is removed along with the -NH2 and the core is metabolized in the usual fashion. If the methyl is on a ring nitrogen, the compound is excreted unchanged in the urine.
Bases to Uric Acid
Both adenine and guanine nucleotides converge at the common intermediate xanthine. Hypoxanthine, representing the original adenine, is oxidized to xanthine by the enzyme xanthine oxidase. Guanine is deaminated, with the amino group released as ammonia, to xanthine. If this process is occurring in tissues other than liver, most of the ammonia will be transported to the liver as glutamine for ultimate excretion as urea.
Xanthine, like hypoxanthine, is oxidized by oxygen and xanthine oxidase with the production of hydrogen peroxide. In man, the urate is excreted and the hydrogen peroxide is degraded by catalase. Xanthine oxidase is present in significant concentration only in liver and intestine. The pathway to the nucleosides, possibly to the free bases, is present in many tissues.
- Uric acid is the end product of purine metabolism.
- Hyperuricaemia is associated with a tendency to form crystals of monosodium urate causing:
- Clinical gout (due to the deposition of monosodium urate crystals in the cartilage, synovium and synovial fluid of joints),
- Renal calculi
- Tophi (accretions of sodium urate in soft tissues)
- Acute urate nephropathy (due to sudden increases in urate production leading to widespread crystallisation in the renal tubules).
Gouts and Hyperuricemia
Both undissociated uric acid and the monosodium salt (primary form in blood) are only sparingly soluble. The limited solubility is not ordinarily a problem in urine unless the urine is very acid or has high [Ca2+]. [Urate salts coprecipitate with calcium salts and can form stones in kidney or bladder.] A very high concentration of urate in the blood leads to a fairly common group of diseases referred to as gout. The incidence of gout in this country is about 3/1000.
Gout is a group of pathological conditions associated with markedly elevated levels of urate in the blood (3-7 mg/dl normal). Hyperuricemia is not always symptomatic, but, in certain individuals, something triggers the deposition of sodium urate crystals in joints and tissues. In addition to the extreme pain accompanying acute attacks, repeated attacks lead to destruction of tissues and severe arthritic-like malformations. The term gout should be restricted to hyperuricemia with the presence of these tophaceous deposits.
Urate in the blood could accumulate either through an overproduction and/or an underexcretion of uric acid. In gouts caused by an overproduction of uric acid, the defects are in the control mechanisms governing the production of - not uric acid itself - but of the nucleotide precursors. The only major control of urate production that we know so far is the availability of substrates (nucleotides, nucleosides or free bases).
One approach to the treatment of gout is the drug allopurinol, an isomer of hypoxanthine.
Allopurinol is a substrate for xanthine oxidase, but the product binds so tightly that the enzyme is now unable to oxidized its normal substrate. Uric acid production is diminished and xanthine and hypoxanthine levels in the blood rise. These are more soluble than urate and are less likely to deposit as crystals in the joints. Another approach is to stimulate the secretion of urate in the urine.
INBORN ERRORS OF PURINE METABOLISM:
A. Hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficiency (Lesch-Nyhan syndrome):
- The Lesch-Nyhan syndrome is an X-linked recessive disorder, due to severe deficiency of HGPRT.
- It is characterised by hyperuricaemia, mental deficiency, spasticity, choreoathetosis and self-mutilation.
- Hyperuricaemia is due to decreased activity of the salvage pathway causing decreased purine reutilization and increased uric acid synthesis. Relatively low levels of nucleotides result in decreased inhibition of de novo synthesis, resulting in further overload of the non-functioning salvage pathway and increased uric acid production.
B. Glucose 6-phosphatase deficiency (Glycogen storage disease type I/ Von Gierke’s disease):
- Deficiency of glucose 6-phosphatase (final enzyme in glycogenolysis pathway) results in accumulation of glycogen, and hypoglycemia.
- Increased metabolism of glucose 6-phosphate through glycolysis results in lactic acidosis.
- Increased metabolism of glucose 6-phosphate through pentose phosphate pathway increases formation of ribose 5-phosphate and NADPH.
- Ribose 5-phosphate is a substrate for increased de novo purine nucleotide synthesis, which is subsequently degraded to uric acid resulting in hyperuricaemia.
- NADPH is a coenzyme in triglyceride synthesis, and overproduction results in hypertriglyceridaemia.
- Hyperuricaemia is aggravated by increased lactic acid which inhibits renal excretion of uric acid.
HG-PRT is deficient in the disease called Lesch-Nyhan Syndrome, a severe neurological disorder whose most blatant clinical manifestation is an uncontrollable self-mutilation. Lesch-Nyhan patients have very high blood uric acid levels because of an essentially uncontrolled de novo synthesis. (It can be as much as 20 times the normal rate). There is a significant increase in PRPP levels in various cells and an inability to maintain levels of IMP and GMP via salvage pathways. Both of these factors could lead to an increase in the activity of the amidotransferase.
Lesch-Nyhan syndrome (LNS) is a rare genetic disorder characterized by an overproduction of uric acid, neurological disability, and behavioral problems. The symptoms of LNS typically appear between ages 3 and 6 months; the presence of orange-colored crystal-like deposits (orange sand) in the child’s diaper is usually the first symptom to appear in those affected with the syndrome.
LNS is caused by a mutation in the HPRT gene on the X-chromosome, resulting in a deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). HPRT is involved in the recycling of purines. When the body is unable to recycle these purines, there is a dramatic overproduction of uric acid, which then leads to hyperuricemia. Hyperuricemia can result in gouty arthritis, tophi (lumpy deposits of uric acid crystals just under the skin) and kidney stones. LNS has been reported to occur in 1 out of every 100,000 live births. It is estimated that there are only several hundred individuals with the disorder in the United States. LNS has been found equally among all races and ethnic groups, however as an X-linked disorder, nearly all cases are male. LNS can either be inherited or it can occur as a spontaneous (or new) mutation.
LNS was first described by Michael Lesch, M.D. and William Nyhan, M.D., Ph.D. in 1964 when they reported two affected brothers. The enzymatic defect was discovered by Seegmiller and colleagues in 1967. Finally, the gene responsible for LNS was cloned and sequenced by Friedmann and colleagues in 1985.
Pyrimidine synthesis begins with carbamoyl phosphate synthesized in the cytosol of those tissues capable of making pyrimidines (highest in spleen, thymus, GItract and testes). This uses a different enzyme than the one involved in urea synthesis. Carbamoyl phosphate synthetase II (CPS II) prefers glutamine to free ammonia and has no requirement for N-Acetylglutamate.
Formation of Orotic Acid
Carbamoyl phosphate condenses with aspartate in the presence of aspartate transcarbamylase to yield N-carbamylaspartate which is then converted to dihydroorotate.
In man, CPSII, asp-transcarbamylase, and dihydroorotase activities are part of a multifunctional protein.
Oxidation of the ring by a complex, poorly understood enzyme produces the free pyrimidine, orotic acid. This enzyme is located on the outer face of the inner mitochondrial membrane, in contrast to the other enzymes which are cytosolic. Note the contrast with purine synthesis in which a nucleotide is formed first while pyrimidines are first synthesized as the free base.
Formation of the Nucleotides
Orotic acid is converted to its nucleotide with PRPP. OMP is then converted sequentially - not in a branched pathway - to the other pyrimidine nucleotides.
Decarboxylation of OMP gives UMP. O-PRT and OMP decarboxylase are also a multifunctional protein. After conversion of UMP to the triphosphate, the amide of glutamine is added, at the expense of ATP, to yield CTP.
The control of pyrimidine nucleotide synthesis in man is exerted primarily at the level of cytoplasmic CPS II. UTP inhibits the enzyme, competitively with ATP. PRPP activates it. Other secondary sites of control also exist (e.g. OMP decarboxylase is inhibited by UMP and CMP). These are probably not very important under normal circumstances.
In bacteria, aspartate transcarbamylase is the control enzyme. There is only one carbamoyl phosphate synthetase in bacteria since they do not have mitochondria. Carbamoyl phosphate, thus, participates in a branched pathway in these organisms that leads to either pyrimidine nucleotides or arginine.
Orotic aciduria refers to an excessive excretion of orotic acid in urine. It causes a characteristic form of anemia and may be associated with mental and physical retardation.
In addition to the characteristic excessive orotic acid in the urine, patients typically have megaloblastic anemia which cannot be cured by administration of vitamin B12 or folic acid.
It also can cause inhibition of RNA and DNA synthesis and failure to thrive. This can lead to mental and physical retardation.
Its hereditary form, an autosomal recessive disorder, can be caused by a deficiency in the enzyme UMPS, a bifunctional protein that includes the enzyme activities of orotate phosphoribosyltransferase and orotidine 5'-phosphate decarboxylase.
It can also arise secondary to blockage of the urea cycle, particularly in ornithine transcarbamylase deficiency (or OTC deficiency). You can distinguish this increase in orotic acid secondary to OTC deficiency from hereditary orotic aciduria (seen above) by looking at blood ammonia levels and the BUN. In OTC deficiency, because the urea cycle backs up, you will see hyperammonemia and a decreased BUN.
Administration of cytidine monophosphate and uridine monophosphate reduces urinary orotic acid and the anemia.
Administration of uridine, which is converted to UMP, will bypass the metabolic block and provide the body with a source of pyrimidine.
In contrast to purines, pyrimidines undergo ring cleavage and the usual end products of catabolism are beta-amino acids plus ammonia and carbon dioxide. Pyrimidines from nucleic acids or the energy pool are acted upon by nucleotidases and pyrimidine nucleoside phosphorylase to yield the free bases. The 4-amino group of both cytosine and 5-methyl cytosine is released as ammonia.
Formation of Deoxyribonucleotides
De novo synthesis and most of the salvage pathways involve the ribonucleotides. (Exception is the small amount of salvage of thymine indicated above.) Deoxyribonucleotides for DNA synthesis are formed from the ribonucleotide diphosphates (in mammals and E. coli).
A base diphosphate (BDP) is reduced at the 2' position of the ribose portion using the protein, thioredoxin and the enzyme nucleoside diphosphate reductase. Thioredoxin has two sulfhydryl groups which are oxidized to a disulfide bond during the process. In order to restore the thioredoxin to its reduced for so that it can be reused, thioredoxin reductase and NADPH are required.
This system is very tightly controlled by a variety of allosteric effectors. dATP is a general inhibitor for all substrates and ATP an activator. Each substrate then has a specific positive effector (a BTP or dBTP). The result is a maintenance of an appropriate balance of the deoxynucleotides for DNA synthesis.