Nucleotides are most often thought of as the building blocks of the nucleic acids, DNA and RNA. While this, is, of course, a vital function, nucleotides also play other important roles in cells. Ribonucleoside triphosphates like ATP, CTP, GTP and UTP are necessary, not just for the synthesis of RNA, but as part of activated intermediates like UDP-glucose in biosynthetic pathways. ATP is also the universal “energy currency” of cells, and coupling of energetically unfavorable reactions with the hydrolysis of ATP makes possible the many reactions in our cells that require an input of energy. Adenine nucleotides serve as components of NAD(P)+ and FAD. Nucleotides can also serve as allosteric and metabolic regulators. The synthesis and breakdown pathways for nucleotides and the molecules derived from them are thus, of vital importance to cells. Regulation of nucleotide synthesis, especially for deoxyribonucleotides, is important to ensure that the four nucleotides are made in the right proportions, as imbalances in nucleotide concentrations can lead to increases in mutation rates.
Pathways of nucleotide metabolism are organized in two major groups and one minor one. These include, respectively, metabolism of 1) purines; 2) pyrimidines; and 3) deoxyribonucleotides. Each group can be further subdivided into pathways that make nucleotides from simple precursors (de novo pathways) and others that use pieces of nucleotides to reassemble full ones (salvage pathways). Notably, de novo synthesis pathways for all of the nucleotides begin with synthesis of ribonucleotides. Deoxyribonucleotides are made from the ribonucleotides.
Synthesis of purine nucleotides by the de novo pathway begins with addition of a pyrophosphate to carbon 1 of ribose-5-phosphate, creating phosphoribosylpyrophosphate (PRPP). The reaction is catalyzed by PRPP synthetase. Some number the purine metabolic pathway starting with the next reaction. We have therefore given this reaction the number of zero in Figure 6.172.
In the next step (reaction 1 in Figure 6.172), the pyrophosphate is replaced by an amine from glutamine in a reaction catalyzed by PRPP amidotransferase (PPAT). The product is 5-phosphoribosylamine (5-PRA).
PPAT is an important regulatory enzyme for purine biosynthesis. The end products of the pathway, AMP and GMP both inhibit the enzyme and PRPP activates it. Interestingly, full inhibition of the enzyme requires binding of both AMP and GMP.
Binding of only one of the two nucleotides allows the enzyme to remain partially active so that the missing nucleotide can be synthesized. Through this enzyme, the relative amounts of ATP and GTP are controlled.
5-PRA is very unstable chemically (half-life of 38 seconds at 37°C), so it has been proposed that it is shuttled directly from PRPP amidotransferase to GAR synthetase for the next reaction.
In this reaction (#2), glycine is added to the growing structure above the ribose-5-phosphate to create glycineamide ribonucleotide (GAR). This reaction, which requires ATP, is catalyzed, as noted, by the enzyme GAR synthetase.
In reaction #3, a formyl group is transferred onto the GAR from N10-formyl-tetrahydrofolate (N10-formyl-THF or fTHF) by phosphoribosylglycinamide formyltransferase (GART).
Next, the double bonded oxygen in the ring is replaced with an amine in a reaction catalyzed by phosphoribosylformylglycinamidine synthase (PFAS) that uses glutamine and produces glutamate. The reaction requires energy from ATP (top of next column).
In humans the GAR synthetase, phosphoribosylglycinamide formyltransferase, and the enzyme catalyzing the next reaction (#5), AIR synthetase activities are all on the same protein known as trifunctional purine biosynthetic protein adenosine-3.
In reaction #6, carboxylation of AIR occurs, catalyzed by phosphoribosylaminoimidazole carboxylase (PAIC)
Aspartic acid is then added to donate its amine group and fumarate will be lost in the reaction that follows this one. The enzyme involved here is phosphoribosyl-aminoimidazole-succinocarboxamide synthase (PAICS)
In the next reaction, the carbon shell of aspartate is released (as fumarate) and the amine is left behind. The reaction is catalyzed by adenylosuccinate lyase (ADSL).
Reaction #9 involves another formylation reaction, catalyzed by phosphoribosylaminoimidazolecarboxamide formyltransferase (ATIC-E1).
Next, inosine monophosphate synthase (ATIC-E2) catalyzes release of water to form the first molecule classified as a purine - inosine monophosphate or IMP).
Though it doesn’t appear in DNA, IMP does, in fact, occur in the anticodon of many tRNAs where its ability to pair with numerous bases is valuable in reading the genetic code.
IMP is a branch point between pathways that lead to GMP or AMP. The pathway to GMP proceeds via catalysis by IMP dehydrogenase as follows:
In the last step of GMP synthesis, GMP synthase catalyzes a transamination to form GMP using energy from ATP.
The energy source being ATP makes sense, since the cell is presumably making GMP because it needs guanine nucleotides. If the cell is low on guanine nucleotides, GTP would be in short supply.
Synthesis of AMP from IMP follows. First, adenylosuccinate synthetase catalyzes the addition of aspartate to IMP, using energy from GTP.
Then, adenylosuccinate lyase splits fumarate off to yield AMP.
In humans, the bifunctional purine biosynthesis protein known as PURH contains activities of the last two enzymes above.
It is worth repeating that synthesis of GMP from IMP requires energy from ATP and that synthesis of AMP from IMP requires energy from GTP. In addition, the enzymes converting IMP into intermediates in the AMP and GMP pathways are each feedback inhibited by the respective monophosphate nucleotide. Thus, IMP dehydrogenase is inhibited by GMP (end product of pathway branch) and adenylosuccinate synthetase is inhibited by AMP, the end product of that pathway branch.
Purine nucleotide levels are balanced by the combined regulation of PRPP amidotransferase , IMP dehydrogenase, adenylosuccinate synthetase and the nucleotides AMP and GMP. The importance of the regulatory scheme of purines is illustrated by two examples. First imagine both AMP and GMP are abundant. When this occurs, PRPP amidotransferase will be completely inhibited and no purine synthesis will occur.
High levels of GMP and low levels of AMP would result in PRPP amidotransferase being slightly active, due to the fact GMP will fill one allosteric site, but low AMP levels will mean second allosteric site will likely be unfilled. This lowered (but not completely inhibited) activity of PRPP amidotransferase will allow for limited production of 5-PRA and the rest of the pathway intermediates, so it will remain active.
At the IMP branch, however, the high levels of GMP will inhibit IMP dehydrogenase, thus shutting off that branch and allowing all of the intermediates to be funneled into making AMP. When the AMP level rises high enough, AMP binds to PRPP amidotransferase and along with GMP, shuts off the enzyme. A reversal will occur if AMP levels are high, but GMP levels are low.
Regulated in this way, AMP and GMP levels can be maintained in a fairly narrow concentration range. Properly balancing nucleotide levels in cells is critical. It is likely for this reason that cells have numerous controls on the amount of each nucleotide made.
Cells have two other ways of balancing GMP and AMP nucleotides. First, the enzyme GMP reductase will convert GMP back to IMP using electrons from NADPH.
The IMP, in turn, can then be made into AMP if its concentration is low. Second, AMP can be converted back to IMP by the enzyme AMP deaminase. In this case, the IMP can then be made into GMP.
It is important to maintain appropriate proportions of the different nucleotides. Excess or scarcity of any nucleotide of any nucleotide can result in an increased tendency to mutation.
To convert AMP to ATP and GMP to GTP requires action of kinase enzymes. Each monophosphate nucleotide form has its own specific nucleoside monophosphate kinase. For adenine-containing nucleotides (ribose forms and deoxyribose forms), adenylate kinase catalyzes the relevant reaction.
The adenylate kinase reaction is reversible and is used to generate ATP when the cell’s ATP concentration is low. When ATP is made from 2 ADPs in this way, AMP levels increase and this is one way the cell senses that it is low on energy.
Guanosine monophosphates also have their own kinase and it catalyzes the reaction at the top of the next page.
Other monophosphate kinases for UMP and CMP use ATP in a similar fashion.
In going from the diphosphate form to the triphosphate form, the picture is simple - one enzyme catalyzes the reaction for all diphosphates (ribose and deoxyribose forms). It is known as nucleoside diphosphate kinase or (more commonly) NDK or NDPK and it catalyzes reactions of the form
where X and Y refer to any base.
Not all nucleotides in a cell are made from scratch. The alternatives to de novo syntheses are salvage pathways. Salvage reactions to make purine nucleotides start with attachment of ribose to purine bases using phosphoribosylpyrophosphate (PRPP).
The enzyme catalyzing this reaction is known as hypoxanthine/guanine phosphoribosyltransferase (HGPRT - Figure 6.175) and is interesting from an enzymological as well as a medical perspective. First, the enzyme is able to catalyze both of the next two important salvage reactions - converting hypoxanthine to IMP or guanine to GMP.
HGPRT is able to bind a variety of substrates at its active site and even appears to bind non-natural substrates, such as acyclovir preferentially over its natural ones.
From a medical perspective, reduction in levels of HGPRT leads to hyperuricemia, a condition where uric acid concentration increases in the body. Complete lack of HGPRT is linked to Lesch-Nyhan syndrome, a rare, inherited disease in high uric acid concentration throughout the body is associated with severe accompanying neurological disorders.
Reduced production of HGPRT occurs frequently in males and has a smaller consequence (gout) than complete absence. Interestingly, gout has been linked to a decreased likelihood of contracting multiple sclerosis, suggesting uric acid may help prevent or ameliorate the disease.
Expression of HGPRT is stimulated by HIF-1, a transcription factor made in tissues when oxygen is limiting, suggesting a role for HGPRT under these conditions.
The enzyme known as adenine phosphoribosyltransferase (APRT) catalyzes the reaction corresponding to HGPRT for salvaging adenine bases.
The de novo pathway for synthesizing pyrimidine nucleotides has about the same number of reactions as the purine pathway, but also has a different strategy. Whereas the purines were synthesized attached to the ribose sugar, pyrimidine bases are made apart from the ribose and then attached later.
The first reaction is catalyzed by carbamoyl phosphate synthetase (Figure 6.176).
Two different forms are found in eukaryotic cells. Form I is found in mitochondria and form II is in the cytoplasm.
The reaction catalyzed by carbamoyl phosphate synthetase is the rate limiting step in pyrimidine biosynthesis and corresponds to reaction 1 in Figure 6.178.
The enzyme is activated by ATP and PRPP and is inhibited by UMP. This helps to balance pyrimidine vs. purine concentrations. High concentrations of a purine (ATP) activates the synthesis of pyrimidines. PRPP increases in concentration as purine concentration increases, so it too helps to establish that balance. UMP is an end product of pyrimidine metabolism, so the process is self-limiting. The next enzyme in the pathway, aspartate transcarbamoylase (ATCase) also plays a role in the same balance, as we will see. The reaction it catalyzes is shown below and is reaction 2 in Figure 6.178.
ATCase is a classic enzyme exhibiting allosteric regulation and feedback inhibition, having both homotropic and heterotropic effectors (Figure 6.179 and see HERE). With 12 subunits (6 regulatory and 6 catalytic units), the enzyme exists in two states - a low activity T-state and a high activity R-state. Binding of the aspartate substrate to the active site shifts the equilibrium in favor of the R-state.
Aspartate is a homotropic effector of the enzyme, because it acts allosterically on the enzyme and is a substrate for it as well. Similarly, binding of ATP
