We examine here the biosynthetic pathways of purine andpyrimidine nucleotides and their regulation, the formation of thedeoxynucleotides, and the degradation of purines and pyrimidines to uric acidand urea.
First, there is evidence, especially in thede novo purine pathway, that the enzymes are present as large, multienzymecomplexes in the cell, a recurring theme in our discussion of metabolism.
DNA contains thymine rather than uracil, and the de novo pathway to thymineinvolves only deoxyribonucleotides. The immediate precursor of thymidylate(dTMP) is dUMP. In bacteria, the pathway to dUMP begins with formation of dUTP,either by deamination of dCTP or by phosphorylation of dUDP (Fig. 16–9). ThedUTP is converted to dUMP by a dUTPase. The latterreaction must be efficient to keep dUTP pools low andprevent incorporation of uridylate into DNA.
Conversion of dUMP to dTMP is catalyzed by thymidylatesynthase. A one-carbon unit at the hydroxymethyl(—CH2OH) oxidation level is transferred fromN5,N10-methylenetetrahydrofolate to dUMP, then reduced to a methyl group (Fig.16–10).The reduction occurs at the expense of oxidation oftetrahydrofolate to dihydrofolate, which is unusual intetrahydrofolate-requiring reactions. The dihydrofolateis reduced to tetrahydrofolate by dihydrofolatereductase—a regeneration that is essential for themany processes that require tetrahydrofolate. In plantsand at least one protist, thymidylate synthase and dihydrofolatereductase reside on a single bifunctional protein.
What's really interesting here is that the ribose sugar is recycled in theform of ribose-1-phosphate, which can be incorporated into PRPP which, as we nowknow, is integral to the biosynthesis of purines, pyrimidines, histidine andtryptophan. That's a really efficient way to run a cell!
We expected that the first step, in which PRPP is synthesized, would besubject to regulation because of the prominence of PRPP in other biosyntheticreactions, including that of pyrimidine nucleotides. Increasing levels of ADPand GDP have a negative feedback effect on the enzyme Ribose phosphatepyrophosphokinase. The enzyme catalyzing the second step of the pathway,Amidophosphoribosyl transferase, is inhibited by all of the adenine and guaninenucleotides, the adenine nucleotides binding to one inhibitory site on theenzyme and the guanine nucleotides to another separate site. This enzyme is also"activated" by the increase in the level of PRPP and this is called a"feedforward activation".
Scheme of the purine metabolismpathways, showing the position of IMPDH2, APRT and PNP in purinenucleotide biosynthesis, adopted from a previous study (35). The de novo synthesis ofpurine nucleotides begins with the phosphorylation ofribose-5-phosphate to form PRPP. In a number of reactions, PRPPcreates the first fully formed nucleotide, IMP. IMP is converted byIMPDH2 to GMP. PNP catalyzes the reversible cleavage of purinenucleosides, releasing purine nucleobases (adenine, hypoxanthine,xanthine and guanine). In the salvage pathway the free nucleobasescan be reconverted back to nucleoside-5′-monophosphates in areaction with activated sugar (PRPP) catalyzed by APRT. IMPDH2,inosine-5′-monophosphate dehydrogenase 2; APRT, adeninephosphoribosyltransferase; PNP, purine nucleoside phosphorylase;PRPP, 5-phosphoribosyl-1-pyrophosphate; IMP,inosine-5′-monophosphate; GMP, guanosine-5′-monophosphate; dADP,deoxyadenosine diphosphate; ADP, adenosine diphosphate; GDP,guanosine diphosphate; dGDP, deoxyguanosine diphosphate; AMP,adenosine monophosphate; XMP, xanthosine monophosphate.
The basic idea here is that there is exquisite control of the amounts ofpurine nucleotides available for synthesis of nucleic acids, and that thepathways are individually regulated at the cellular level. Furthermore, therelative amounts of ATP and GTP are also controlled at the cellular level.
The downregulation of the 3 key enzymes of purinemetabolism can have a profound effect on nucleotide homeostasis inTRAIL-resistant lymphoma cells. Purine nucleotides, the buildingblocks for synthesis of DNA, RNA and enzyme co-factors, arerecruited either from purine synthesis from lowmolecular weight precursors or by recycling of free nucleobases inthe so-called salvage pathway. Both pathways lead to the productionof nucleoside-5′-phosphates (). Both pathways can supply cellular demand independently;however, their importance in different tissues is variable. Inleukemic and lymphoma cells the salvage pathway is considered themajor source of purine nucleotides (,).
When ATP appears as a reactant, it can generally participate in two ways:part of the ATP molecule can be transferred to an acceptor molecule or ATPhydrolysis can drive an otherwise unfavorable reaction. The Pi, PPi,adenyl or adenosinyl groups can be transferred, as in the first step in thepurine biosynthetic pathway. In such instances, the substrate is said to be"activated" by the transfer. When the free energy of ATP hydrolysisdrives an endergonic reaction, the overall mechanism must involve transfer of aPi group somewhere along the way, even though, in the final analysis,it will appear as Pi in the reaction ATP + H20 -->ADP + Pi. Otherwise, there would be no way to couple thereactions.
The synthesis of purine nucleotidesrequires 5-phosphoribosyl-1-pyrophosphate (PRPP), ATP, glutamine,glycine, CO, aspartate and formate to create the firstfully formed nucleotide, inosine-5′-monophosphate (IMP). IMPrepresents a branch point for purine biosynthesis, since it can beconverted either to guanosine-5′-monophosphate (GMP) by IMPDH2(downregulated in HBL-2/R cells) or to adenosine-5′-monophosphate().
The 5-Phosphoribosyl--pyrophosphate (PRPP) formedin the first step is also a precursor of pyrimidine synthesis, and it is also aprecursor of the synthesis of Trp and His. Because it is part of so manyimportant pathways, it is highly regulated. The role of ATP in this step isdifferent from that of the other steps in this pathway in which it is found. ATPactivates the ribose-5-phosphate by adding a pyrophosphate group (PPi)to C1 of the sugar (i.e., there is a group transfer). All of the otherATP-involving steps that follow harness the energy of hydrolysis of a phosphatebond of ATP (exergonic) to drive an endergonic reaction. In these steps, one cantalk about "coupling" of two reactions, such that the exergonic onedrives the endergonic one with the result that the overall G'is negative.
The delicate balance of enzyme activities andconcentrations of products and intermediates are critical forpurine (nucleotide) homeostasis. The inhibition of PNP results inthe accumulation of its substrate, 2′-deoxyguanosine which isfurther phosphorylated to deoxyguanosine triphosphate (dGTP). Ahigh intracellular concentration of dGTP inhibits cellproliferation and induces apoptosis (–). If APRT is inhibited, accumulatedadenine is oxidized to insoluble 2,8-dihydroxyadenine. Accumulationof this precipitate results in cell death (). Similarly, the inhibition of IMPDH2leads to depletion of guanosine nucleotides, which blocks DNAsynthesis and cell division (,).