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ThePurine Nucleotide Cycle is ametabolic pathway inprotein metabolism requiring the amino acidsaspartate andglutamate. The cycle is used to regulate the levels ofadenine nucleotides, in whichammonia andfumarate are generated.[2]AMP converts intoIMP and the byproduct ammonia. IMP converts to S-AMP (adenylosuccinate), which then converts to AMP and the byproduct fumarate. The fumarate goes on to produce ATP (energy) viaoxidative phosphorylation as it enters theKrebs cycle and then theelectron transport chain. Lowenstein first described this pathway and outlined its importance in processes includingamino acid catabolism and regulation of flux throughglycolysis and theKrebs cycle.[2][3][4]
AMP is produced after strenuous muscle contraction when the ATP reservoir is low (ADP > ATP) by theadenylate kinase (myokinase) reaction.[5][6] AMP is also produced from adenine and adenosine directly; however, AMP can be produced through less direct metabolic pathways, such asde novo synthesis of IMP or through salvage pathways ofguanine (a purine) and any of thepurine nucleotides and nucleosides. IMP is synthesizedde novo fromglucose through thepentose phosphate pathway which producesribose 5-P, which then converts toPRPP that with the amino acids glycine, glutamine, and aspartate (seePurine metabolism) can be further converted into IMP.[7]
The cycle comprises threeenzyme-catalysed reactions. The first stage is the deamination of the purinenucleotideadenosine monophosphate (AMP) to forminosine monophosphate (IMP), catalysed by the enzymeAMP deaminase:
The second stage is the formation ofadenylosuccinate from IMP and the amino acidaspartate, which is coupled to the energetically favourable hydrolysis ofGTP, and catalysed by the enzymeadenylosuccinate synthetase:
Finally, adenylosuccinate is cleaved by the enzymeadenylosuccinate lyase to release fumarate and regenerate the starting material of AMP:
A recent study showed that activation of HIF-1α allows cardiomyocytes to sustain mitochondrial membrane potential during anoxic stress by utilizing fumarate produced by adenylosuccinate lyase as an alternate terminal electron acceptor in place of oxygen. This mechanism should help provide protection in the ischemic heart.[8]
The purine nucleotide cycle occurs in thecytosol (intracellular fluid) of thesarcoplasm ofskeletal muscle, and in themyocyte'scytosolic compartment of thecytoplasm ofcardiac andsmooth muscle. The cycle occurs when ATP reservoirs run low (ADP > ATP), such as strenuous exercise, fasting or starvation.[5][9]
Proteins catabolize into amino acids, andamino acids are precursors for purines, nucleotides and nucleosides which are used in the purine nucleotide cycle.[7] The amino acidglutamate is used to neutralize the ammonia produced when AMP is converted into IMP. Another amino acid,aspartate, is used along with IMP to produce S-AMP in the cycle. Skeletal muscle contains amino acids for use in catabolism, known as the free amino acid pool; however, inadequate carbohydrate supply and/or strenuous exercise requires protein catabolism to sustain the free amino acids.[9]
When thephosphagen system (ATP-PCr) has been depleted ofphosphocreatine (creatine phosphate), the purine nucleotide cycle also helps to sustain themyokinase reaction by reducing accumulation of AMP produced after muscle contraction in the below reaction.[6]
During muscle contraction:
Muscle at rest:
AMP can dephosphorylate to adenosine anddiffuse out of the cell; the purine nucleotide cycle may therefore also reduce the loss of adenosine from the cell since nucleosides permeate cell membranes, whereas nucleotides do not.[6]
Fumarate, produced from the purine nucleotide cycle, is an intermediate ofTCA cycle and enters themitochondria by converting into malate and utilizing themalate shuttle where it is converted into oxaloacetic acid (OAA). During exercise, OAA either enters intoTCA cycle or converts into aspartate in the mitochondria.[10]
As the purine nucleotide cycle produces ammonia(see below in ammonia synthesis), skeletal muscle needs to synthesize glutamate in a way that does not further increase ammonia, and as such the use ofglutaminase to produce glutamate from glutamine would not be ideal. Also, plasma glutamine (released from the kidneys) requiresactive transport into the muscle cell (consuming ATP).[11] Consequently, during exercise when the ATP reservoir is low (ADP>ATP), glutamate is produced from branch-chained amino acids (BCAAs) and α-ketoglutarate, as well as from alanine and α-ketoglutarate.[12] Glutamate is then used to produce aspartate. The aspartate enters the purine nucleotide cycle, where it is used to convert IMP into S-AMP.[10][13]
When skeletal muscle is at rest (ADP<ATP), the aspartate is no longer needed for the purine nucleotide cycle and can therefore be used with α-ketoglutarate to produce glutamate and oxaloacetic acid (the above reaction reversed).
α-Ketoglutarate + Aspartate ⇌ Oxaloacetic acid + Glutamate (catalyzed byaspartate aminotransferase)
During exercise when the ATP reservoir is low (ADP>ATP), the purine nucleotide cycle produces ammonia (NH3) when it converts AMP into IMP. (With the exception ofAMP deaminase deficiency, where ammonia is produced during exercise when adenosine, from AMP, is converted into inosine). During rest (ADP<ATP), ammonia is produced from the conversion of adenosine into inosine by adenosine deaminase.
Ammonia is toxic, disrupts cell function, and permeates cell membranes. Ammonia becomes ammonium (NH+
4) depending on the pH of the cell or plasma. Ammonium is relatively non-toxic and does not readily permeate cell membranes.[14]
NH3 +H+
⇌NH+
4
Ammonia (NH3) diffuses into the blood, circulating to the liver to be neutralized by theurea cycle. (N.b.urea is not the same asuric acid, though both are end products of the purine nucleotide cycle, from ammonia and nucleotides respectively.) When the skeletal muscles are at rest (ADP<ATP), ammonia (NH3) combines with glutamate to produceglutamine, which is an energy-consuming step, and the glutamine enters the blood.[15][11]
Glutamate +NH3 + ATP → Glutamine + ADP +P
i (catalyzed byglutamine synthetase in resting skeletal muscle)
Excess glutamine is used byproximal tubule in the kidneys for ammoniagenesis, which may counteract any metabolic acidosis from anaerobic skeletal muscle activity.[15] In kidneys, glutamine is deaminated twice to form glutamate and thenα-ketoglutarate. TheseNH3 molecules neutralise the organic acids (lactic acid andketone bodies) produced in the muscles.
Glutamine +H2O → Glutamate +NH+
4 (catalyzed byglutaminase in the kidneys)
Somemetabolic myopathies involve the under- or over-utilization of the purine nucleotide cycle. Metabolic myopathies cause a low ATP reservoir in muscle cells (ADP > ATP), resulting in exercise-induced excessive AMP buildup in muscle, and subsequent exercise-induced hyperuricemia (myogenic hyperuricemia) through conversion of excessive AMP into uric acid by way of either AMP → adenosine or AMP → IMP.
During strenuous exercise, AMP is created through the use of the adenylate kinase (myokinase) reaction after the phosphagen system has been depleted of creatine phosphate and not enough ATP is being produced yet by other pathways(see above reaction in 'Occurrence' section). In those affected by metabolic myopathies, exercise that normally wouldn't be considered strenuous for healthy people, is however strenuous for them due to their low ATP reservoir in muscle cells. This results in regular use of the myokinase reaction for normal, everyday activities.
Besides the myokinase reaction, a high ATP consumption and low ATP reservoir also increases protein catabolism and salvage of IMP, which results in increased AMP and IMP. These two nucleotides can then enter the purine nucleotide cycle to produce fumarate which will then produce ATP by oxidative phosphorylation. If the purine nucleotide cycle is blocked (such as AMP deaminase deficiency) or if exercise is stopped and increased fumarate production is no longer needed, then the excess nucleotides will be converted into uric acid.
AMP deaminase deficiency (formally known as myoadenylate deaminase deficiency or MADD) is ametabolic myopathy which results in excessive AMP buildup brought on by exercise. AMP deaminase is needed to convert AMP into IMP in the purine nucleotide cycle. Without this enzyme, the excessive AMP buildup is initially due to the adenylate kinase (myokinase) reaction which occurs after a muscle contraction.[16] However, AMP is also used to allosterically regulate the enzymemyophosphorylase (seeGlycogen phosphorylase § Regulation), so the initial buildup of AMP triggers the enzyme myophosphorylase to release muscle glycogen into glucose-1-P (glycogen→glucose-1-P),[17] which eventually depletes the muscle glycogen, which in turn triggers protein metabolism, which then produces even more AMP. In AMP deaminase deficiency, excessadenosine is converted into uric acid in the following reaction:
Myogenichyperuricemia, as a result of the purine nucleotide cycle running when ATP reservoirs in muscle cells are low (ADP > ATP), is a common pathophysiologic feature ofglycogenoses such asGSD-III,GSD-V andGSD-VII, as they aremetabolic myopathies which impair the ability of ATP (energy) production within muscle cells. In these metabolic myopathies, myogenic hyperuricemia is exercise-induced; inosine, hypoxanthine and uric acid increase in plasma after exercise and decrease over hours with rest.[18] ExcessAMP (adenosine monophosphate) is converted intouric acid.[18]
Hyperammonemia is also seen post-exercise in McArdle disease (GSD-V) andphosphoglucomutase deficiency (PGM1-CDG, formerly GSD-XIV), due to the purine nucleotide cycle running when the ATP reservoir is low due to the glycolytic block.[19][20][21][22][23][24]
AMP +H2O +H+
→ IMP +NH3