S-adenosylmethionine (SAMe) appears to partially alleviate purine depletion via a PRPP-independent path. Synthesis of pyrimidine nucleotides is PRPP dependent, with uridine monophosphate synthase deficiency producing pyrimidine nucleotide depletion.
Full Answer
What is the substrate of PRPP synthetase?
PRPP synthetase has an absolute requirement for inorganic phosphate (P i ), which functions as an allosteric activator. The enzyme is inhibited by many nucleotides, the end products of the pathway for which PRPP is an essential substrate. The gene for PRPP synthetase is located on the X-chromosome.
Are allosteric variants of PRS-I superactivity associated with defective enzyme regulation?
All of the pathogenic variants identified to date in individuals with the PRS superactivity phenotype have resulted in defective allosteric regulation of PRS-I enzyme activity; however, this finding is biased by the fact that pathogenic variants were sought on the basis of metabolic and neurodevelopmental phenotypes.
What is the role of PRPP in DNA synthesis?
5-Phosphoribosyl-1-pyrophosphate (PRPP) is a key intermediate in nucleotide biosynthesis. It is required for de novo synthesis of purine and pyrimidine nucleotides and the salvage pathways, in which purines are converted to their respective nucleotides via transfer of ribose 1-phosphate group from PRPP to the base, i.e.:
How is PRPP synthesized from ribose 5 phosphate?
PRPP is synthesized from ribose 5-phosphate by the following reaction: In this reaction, the pyrophosphate group of ATP is transferred to ribose 5-phosphate; the product PRPP is a high-energy compound. PRPP synthetase has an absolute requirement for inorganic phosphate (Pi), which functions as an allosteric activator.
What causes PRPP synthetase deficiency?
Inheritance. This condition is inherited in an X-linked pattern. The gene associated with this condition is located on the X chromosome, which is one of the two sex chromosomes . In males (who have only one X chromosome), a mutation in the only copy of the gene in each cell causes the disorder.
What is PRPP synthetase inhibited by?
PRPP synthetase has an absolute requirement for inorganic phosphate (Pi), which functions as an allosteric activator. The enzyme is inhibited by many nucleotides, the end products of the pathway for which PRPP is an essential substrate. The gene for PRPP synthetase is located on the X-chromosome.
How is PRPP synthase regulated?
Non-oxidative pentose phosphate pathway enzymes showed no change in activity, but PRPP synthetase is regulated by phosphate, and we found that phosphate uptake and total intracellular phosphate concentration increased significantly between mid-G1-phase and early S-phase.
What activates PRPP synthetase?
PRS catalyzes the PRPP synthesis from Mg-ATP and ribose-5-phosphate in a reaction that requires Mg2+ and inorganic phosphate (Pi) as activators.
What enzyme deficiency causes gout?
Hypoxanthine:guanine phosphoribosyltransferase deficiency is known to cause hyperuricaemia and gout.
What is the importance of PRPP?
Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds.
What are the substrates of PRPP synthetase?
Phosphoribosylpyrophosphate (PRPP) is a substrate involved in the synthesis of essentially all nucleotides and important in the regulation of the de novo pathways of purine and pyrimidine nucleotide synthesis. The synthetase enzyme (PRPS) produces PRPP from ribose-5-phosphate and ATP (seeFigs.
How is purine synthesis regulated?
Purine nucleotide biosynthesis is regulated at several steps IMP synthesis is controlled by the levels of adenine and guanine nucleotides. Additional control is exerted by feedforward activation, which is the stimulation of a subsequent enzyme by the preceding substrate.
What enzyme makes PRPP from ribose-5-phosphate?
Ribose phosphate pyrophosphokinase catalyzes the transfer of pyrophosphate from ATP to ribose 5-phosphate to form AMP and phosphoribosyl pyrophosphate (PRPP).
What is PRPP in DNA?
Phosphoribosyl pyrophosphate (PRPP) is a pentose phosphate and a biochemical intermediate in the formation of purine nucleotides via inosine-5-monophosphate. Hence it is a building block for DNA and RNA. The vitamins thiamine and cobalamin, and the amino acid tryptophan also contain fragments derived from PRPP.
Is PRPP a cofactor?
PRPP is cofactor for uridine monophosphate synthetase (UMPS), which converts orotic acid into UMP, the precursor of all other pyrimidine nucleotides.
Why is PRPS1 overactive?
PRPS1 gene overactivity increases the production of normal PRPP synthetase 1 enzyme, which increases the availability of PRPP. In both forms of the disorder, excessive amounts of purines are generated.
What happens if you have a mutation in PRPS1?
In people with the more severe form of PRS superactivity, PRPS1 gene mutations change single protein building blocks ( amino acids) in the PRPP synthetase 1 enzyme, resulting in a poorly regulated, overactive enzyme. In the milder form of PRS superactivity, the PRPS1 gene is overactive for reasons that are not well understood.
What is the function of PRPS1?
Certain mutations in the PRPS1 gene cause PRS superactivity . The PRPS1 gene provides instructions for making an enzyme called phosphoribosyl pyrophosphate synthetase 1, or PRPP synthetase 1. This enzyme helps produce a molecule called phosphoribosyl pyrophosphate (PRPP). PRPP is involved in producing purine and pyrimidine nucleotides. These nucleotides are building blocks of DNA, its chemical cousin RNA, and molecules such as ATP and GTP that serve as energy sources in the cell. PRPP synthetase 1 and PRPP also play a key role in recycling purines from the breakdown of DNA and RNA, a faster and more efficient way of making purines available.
When does PRS superactivity start?
There are two forms of PRS superactivity, a severe form that begins in infancy or early childhood, and a milder form that typically appears in late adolescence or early adulthood. In both forms, a kidney or bladder stone is often the first symptom.
How many families have PRS superactivity?
PRS superactivity is believed to be a rare disorder. Approximately 30 families with the condition have been reported. More than two thirds of these families are affected by the milder form of the disease.
What is PRPP synthetase?
PRPP synthetase has an absolute requirement for inorganic phosphate (Pi), which functions as an allosteric activator.
Where is PRPP synthetase located?
The gene for PRPP synthetase is located on the X-chromosome. Mutations in this gene have given rise to PRPP synthetase variants with increased catalytic activity, which leads to overproduction of uric acid (discussed later, under “Gout”). The main source of ribose-5-phosphate is the pentose phosphate pathway (Chapter 14 ).
What is the role of PRPP in purine metabolism?
In addition, PRPP also plays an important role in regulating the expression of genes in purine metabolism as discussed later. The compound PRPP is synthesized by PRPP synthase that transfers the β,γ-diphosphoryl group from ATP to the 1-position of ribose 5-phosphate to give PRPP: ATP + Ribose 5 - phosphate → PRPP + AMP.
What is PRPP in the purine pathway?
PRPP is substrate of three enzymes of purine metabolic pathway: PRPP amidotransferase, in de novo synthesis pathway, which serves specifically as the rate-limiting reaction for the purine synthesis, and HPRT and APRT in the salvage pathway.
What is PRPP in biology?
PRPP serves as a cosubstrate for a diverse family of enzymes, only three of which are involved directly in purine metabolism. These include the first and rate-limiting step of de novo purine synthesis (amidophosphoribosyltransferase (AMPRT)) as well two recycling enzymes (APRT and HPRT).
What is the name of the gene that increases the activity of PRPP synthase?
Mutations in the PRPS1 gene that increase the activity of PRPP synthase lead to sensorineural hearing loss.
What is the role of PRPP in nucleotide biosynthesis?
It is required for de novo synthesis of purine and pyrimidine nucleotides and the salvage pathways, in which purines are converted to their respective nucleotides via transfer of ribose 1-phosphate group from PRPP to the base , i.e.:
What is the function of PRPP synthetase?
PRPP synthetase is an important enzyme that produces a key metabolic intermediate necessary for the biosynthesis of purine and pyrimidine nucleotides. Dysfunction of this protein in humans can lead to severe neurodevelopmental disability. Herein, we provide the details of a 2.2 Å X-ray crystal structure of the PRPP synthetase from E. coli. Our structure reveals the molecular details of the active site of this hexameric protein and identifies the key residues for substrate binding and catalysis. In addition, the putative allosteric site and flexible loop that defines this region are well resolved in the structure. The flexible loop is found in a position that occupies key binding interactions for the allosteric regulator, thus suggesting a possible mechanism to prevent molecules with low affinity binding to this site. This work expands our understanding of this important class of enzyme and provides a structural framework for future studies.
What is the function of PRPP?
The metabolite phosphoribosylpyrophosphate (PRPP) is a building block for the biosynthesis of purine and pyrimidine nucleotides. Ribose-phosphate diphosphokinases or PRPP synthetases (EC 2.7.6.1, also called ribose-phosphate pyrophosphokinases) are a family of enzymes that catalyze the synthesis of PRPP from ATP and ribose-5-phosphate (R5P), yielding AMP as an additional product. This reaction effectively links the pentose phosphate pathway to the nucleotide salvage and de novo biosynthetic pathways as well as the biosynthesis of histidine, tryptophan, and pyridine nucleotide coenzymes [ 1, 2 ]. As PRPP is a metabolite that is required at all times in cells, the proper function of PRPP synthetases is essential for life. In humans, mutations that cause overactivity of this enzyme lead to excessive production of uric acid, which causes gout, developmental abnormalities and neurological impairment [ 3, 4, 5, 6, 7 ]. Three classes (I, II and III) of PRPP synthetases have been described and are differentiated based on phosphate requirements, allosteric regulatory mechanisms, and specificity [ 8, 9, 10, 11, 12, 13 ]. Several crystal structures have been solved of PRPP synthetases, including those from B. subtilis (class I) [ 8 ], M. jannaschii (Class III), [ 10 ], B. pseudomallei (class I) [ 14] and human (class I) [ 15 ]. The E. coli ribose-phosphate pyrophosphokinase (EcKPRS) is a class I PRPP synthetase with well-defined kinetics [ 9, 16, 17, 18 ]. Like other class I PRPP synthetases, EcKPRS is known to bind magnesium for efficient catalysis and makes use of an allosteric regulatory site [ 9, 17, 18 ]. Here, we report the X-ray crystal structure of EcKPRS, determined to 2.2 Å resolution#N#Footnote#N#1. The structure, determined by molecular replacement, confirms the hexameric organization of the protein and provides clear molecular details of both the active site and allosteric regulatory site. This work expands the repertoire of structurally characterized PRPP synthetases and will be a valuable tool to aid further detailed analyses of this important protein.
What is the KPRS gene?
The E. coli (strain K12) ribose-phosphate pyrokinase ( KPRS) gene (UniProt P0A717, NCBI NP_415727.1) was synthesized (GenScript) and cloned as an N-terminally His-tagged protein into the pTHT vector (a modified form of pET-28 with a Tobacco Etch Virus protease site in place of the thrombin site [ 24 ]) and expressed in E coli BL21 (DE3) strain. Bacterial cultures were grown in LB broth supplemented with 50 mg/L kanamycin to an optical density at 600 nm of 0.7, at which point protein expression was induced with 0.1 m M isopropyl thiogalactopyranoside (IPTG). The induced cells were grown, with shaking, at 310 K for another four hours. The cultures were pelleted and the resulting pellets were stored at − 20 °C. For purification, freshly thawed cells were resuspended and lysed with lysis buffer (3 mL/g of pellet; 50 mM phosphate pH 7.6, 300 mM NaCl, 10 mM Imidazole). Cell lysates were passed over a pre-equilibrated (lysis buffer) Ni-NTA column (Qiagen). Non-specifically bound proteins were eluted with lysis buffer supplemented with 15 mM imidazole. KPRS protein was eluted using with lysis buffer supplemented with 250 mM imidazole. The protein was buffer exchanged using a 10DG column (BioRad) to storage buffer containing 10 mM Tris-HCl pH 7.6 and 30 mM NaCl, before concentrating the protein to a final concentration of 10 mg/mL as determined by absorbance on a NanoDrop (ThermoFisher). The protein was determined to be greater than 90% pure by SDS-PAGE. The final protein stock was aliquoted, flash frozen in liquid nitrogen, and stored at − 80 °C.
What is ribose phosphate pyrophosphokinase?
Ribose-phosphate pyrophosphokinase (EC 2.7.6.1) is an enzyme that catalyzes the ATP-dependent conversion of ribose-5-phosphate to phosphoribosyl pyrophosphate. The reaction product is a key precursor for the biosynthesis of purine and pyrimidine nucleotides.
What is the key structural feature of an enzyme that catalyzes a critical step in nucleot
Overall, this study details key structural features of an enzyme that catalyzes a critical step in nucleotide metabolism. This work provides a framework for future studies of this important protein and, as nucleotides are critical for viability, may serve as a foundation for the development of novel anti-bacterial drugs.
What is PRPP used for?
PRPP plays a role in purine salvage pathways by reacting with free purine bases to form adenylate, guanylate, and inosinate. PRPP is also used in the synthesis of NAD: the reaction of PRPP with nicotinic acid yields the intermediate nicotinic acid mononucleotide.
What is the function of PRPP?
Function. The product of this reaction, phosphoribosyl pyrophosphate (PRPP), is used in numerous biosynthesis ( de novo and salvage) pathways. PRPP provides the ribose sugar in de novo synthesis of purines and pyrimidines , used in the nucleotide bases that form RNA and DNA.
What color is the ribose 5-phosphate binding region?
Flexible loop colored in green; ribose 5-phosphate binding region colored in blue. PyMol rendering of phosphoribosyl pyrophosphate synthetase I (human) as a homodimer, formed by two subunits (red and blue). Three homodimers form the active enzyme complex.
How is ribose phosphate diphosphokinase regulated?
Ribose-phosphate diphosphokinase is regulated by phosphorylation and allostery. It is activated by phosphate and inhibited by ADP; it is suggested that phosphate and ADP compete for the same regulatory site. At normal concentrations, phosphate activates the enzyme by binding to its allosteric regulatory site.
What is the enzymatic reaction of ribose phosphate?
The enzymatic reaction begins with the binding of ribose 5-phosphate, followed by binding of Mg-ATP to the enzyme. In the transition state upon binding of both substrates, the diphosphate is transferred. The enzyme first releases AMP before releasing the product phosphoribosyl pyrophosphate. Experiments using oxygen 18 labelled water demonstrate that the reaction mechanism proceeds with the nucleophilic attack of the anomeric hydroxyl group of ribose 5-phosphate on the beta-phosphorus of ATP in an SN2 reaction.
What is the reaction between PRPP and glutamine?
The reaction of PRPP, glutamine, and ammonia forms 5-Phosphoribosyl-1-amine, a precursor to inosinate (IMP), which can ultimately be converted to adenosine triphosphate (ATP) or guanosine triphosphate (GTP). PRPP plays a role in purine salvage pathways by reacting with free purine bases to form adenylate, guanylate, and inosinate.
What is ADP inhibitor?
ADP is the key allosteric inhibitor of ribose-phosphate diphosphokinase. It has been shown that at lower concentrations of the substrate ribose 5-phosphate, ADP may inhibit the enzyme competitively. Ribose-phosphate pyrophosphokinase is also inhibited by some of its downstream biosynthetic products.
