Phosphoribosyl Pyrophosphate Synthetase - Essay Example

Introduction: In the 1850s, Louis Pasteur concluded that fermentation of sugar into alcohol by yeast is catalyzed by "ferments". Then in 1897 Eduard Buchner discovered that yeast extracts could ferment sugar to alcohol providing that fermentation was performed by molecules which continued to function when removed from cells. Fredrick W Kuhne called these molecules enzymes. With the exception of a few catalytic RNA molecules, all enzymes are proteins. Their catalytic activity depends on the integrity of their native conformation. Some enzymes require no chemical groups for activity other than their amino acid residues. Others require an additional chemical component called a cofactor such as an inorganic ion Fe2+, Mg2+, Zn2+ or a metalloorganic molecule called a coenzyme. (Lehninger 4th Edition) Enzymes are classified into six classes each with sub classes based on the type of reaction catalyzed. Thus our enzyme of interest Phosphoribosylpyrophosphate (PRPP) Synthetase (Prs) belongs to the class Transferases catalyzing group transfer reactions with its Enzyme Commission Number as 2.7.6.1. (Online Mendelian Inheritance in Man, 2010) Phosphoribosylpyrophosphate synthetase (Prs) (or Ribose-phosphate diphosphokinase) catalyses the synthesis of phosphoribosyl pyrophosphate (PRPP), an intermediate in nucleotide metabolism for the de novo and salvage pathways of purine and pyrimidine biosynthesis and the biosynthesis of the amino acids histidine and tryptophan. Purine and Pyrimidine Synthesis: One of the important specialized pathways of a number of amino acids is the synthesis of purine and pyrimidine nucleotides. The two ringed purine members Adenine, Guanine, Hypoxanthine and Xanthine and the single ringed pyrimidines namely Cytosine, Thymine, Uracil 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. We can both synthesize them de novo and salvage and reuse those we already have.(Angstadt 1997) Metabolic contribution of PRPP to the purine biosynthesis and other anabolic pathways Source: Taken and Adapted from BMC Biotechnology (Jimenez 2008) PRPP, synthesized from ribose-5-phosphate and ATP, is therefore a key compound for purine biosynthesis, and it is also an important cellular metabolite because it represents a link between carbon and nitrogen metabolism. PRPP is a biosynthetic precursor of histidine and tryptophan, and it is also required for the de novo and salvage pathways of purine, pyrimidine and pyridine (NAD+, NADP+) nucleotides. It has been calculated that approximately 80% of the metabolic flux through PRPP is directed to purine and pyrimidine synthesis The formation of PRPP is catalyzed by the enzyme PRPP synthetase which is encoded by PRS genes Pathway of Purine nucleotide synthesis and its regulation by PRPP. Taken and adapted from (Becker, Kim et al. 1992) The PRPP is further committed to de novo purine nucleotide synthesis in a 10 step enzymatic reaction as follows: Taken From Rolfes 2006 This essay will examine the reactions that take place during the PRPP pathways and the synthesis of purine nucleotide. In microbes such as Escherichia coli and Saccharomyces cerevisiae, the inability to biosynthesize purine nucleotides leads to auxotrophy. In Drosophila, purine nucleotide synthesis is required for development and metamorphosis. In plants such as Arabidopsis and tobacco (Nicotiana tabacum), synthesis of nucleotides is developmentally regulated, whereas in the tropical legumes it plays an additional important role in nitrogen storag. In humans, disorders in the purine nucleotide biosynthetic and salvage pathways have devastating consequences, leading to disorders such as SCIDS (severe combined immunodeficiency syndrome), Lesch-Nyhan syndrome, mental retardation and autism. Also, it has been shown that a reduced expression levels of the phosphoribosyl pyrophosphate synthetase 1 gene (PRPS1), a member of the PRPP synthetase family, results in the Arts syndrome with some of its clinical symptoms covering mental retardation, hypotonia and ataxia(de Brouwer, Williams et al. 2007). De Brouwer found that reduce expression of PRPS1 was due to a missence mutation which has as an effect the malfunction of phosphoribosyl pyrophosphate synthetase 1 (PRS-I) activity. Thus the ability to maintain nucleotide pools is extremely important to cells. By investigating these important pathways, the current essay will give an insight to the therapeutic prospective of purigenic signalling. Bacterial PRPP Because of its key position in nucleotide metabolism PRPP synthase is highly conserved among different organisms. PRPP synthases from the bacteria Bacillus subtilis and Escherichia coli have proven to be good models for the human enzyme. All three are allosterically inhibited by ADP (and GDP) and activated by monophosphate. Allostery means that the site for regulation has a different location on the enzyme than the active site. Phospho ribosyl pyrophosphate (PRPP) synthetase in E.coli was characterized by Jensen in 1985. He observed the following properties. Magnesium ions were required both as a complex with substrate ATP and as free cation essential. PRPP synthetase activity was strongly inhibited by ADP, multiple sites of action were suggested for which. In addition apparent substrate inhibition was exerted by ribose 5-phosphate in the presence of ADP. The pH activity profile of E.coli PRPP synthetase was showed maximal activity at pH 9.7-9.8, whereas the enzyme from S. typhimurium had its optimum at pH 9.2. E. coli PRPP synthetase had no activity when assayed in the absence of Pi as observed by Jensen. Activation by Pi followed a hyperbolic curve with half-maximal activation at 20 mM potassium phosphate. Phosphate was also required to stabilize the enzyme. A concentration of 50 mM potassium phosphate or higher was required to stabilize the enzyme fully. ADP inhibition of E. coli P-Rib-PP synthetase was potent and kinetically complex mostly by means of an allosteric inhibition site. ADP inhibition was noncompetitive with respect to ATP concentration. As with most ATP-dependent enzymes, it was observed that E.coli PRPP synthetase required divalent cations for activity. The most striking feature of the nucleotide sequence, however, is the unusually long distance between the transcription and translation initiation sites. It appears that 302 nucleotides upstream from the presumed translation initiation codon are transcribed. The functioning of this sequence was unknown at the time of this publication. It was postulated to contain sequences involved in regulation of prs expression. It is also noteworthy that translation of Prs is initiated at a GUG codon. According to Kozak (1983), GUG codons are usually used in reinitiation of translation following nonsense codons. The structure and properties of E. coli PRPP synthetase are very similar to the properties of the enzyme from S. typhimurium. However PRPP synthetase is not solely essential in the de novo pathway was further elucidated by Jensen in 2003 where an alternative enzyme system comprising of phosphopentomutase, ribose 1-phosphokinase, and ribose 1,5-bisphosphokinase was discovered by him. The pathway which was analyzed in Escherichia coli led to the synthesis of 5-phospho-D-ribosyl -1-diphosphate (PRPP) without the participation of PRPP synthase. This pathway was revealed by selection for suppression of the NAD requirement of strains with a deletion of the prs gene. Crystal structure of Bacillus subtilis PRPP synthetase coloured by subunit. Taken from Centre for crystallographic Studies, Department of Chemistry, University of Copenhagen Yeast PRPP Synthetase Saccharomyces cerevisiae harbours five unlinked structural genes encoding Prs polypeptides Prs1 to Prs5p. Within this PRS multi gene family, PRS2, PRS3 and PRS4 exhibit more Sequence similarity to each other than to either PRS1 or PRS5 (Carter etal.,1994,1997). Characterization of yeast strains deleted individually for each of the PRS genes indicates that, although none of them is essential for viability, the contribution of each of the gene products to the metabolic status of the cell appears to dier both qualitatively and quantitatively. Disruption of the PSR1 or PSR3 gene results in severly reduced growth rate and substantially low overall enzyme activity suggesting that these polypeptides have a key structural or regulatory role in PRPP synthesis. Immunofluorescence analysis of the subcellular distribution of Prs1p suggested that Prs1p is a cytosolic enzyme predominantly found in granular structures that localize to the cell perimeter, below the plasma membrane in a study by Shcneiter in 2000. This arrangement was confirmed by analyzing the subcellular localization of a functional GFPPrs1p fusion protein. The fact that Prs1p localization is not aected in secretory mutants indicates that the segranular structures do not represent a post Golgi compartment. Prs1p is also shown to exist in an aggregated form or in association with non soluble cytoplasmic structures. The results presented here and elsewhere (Hernando et al.,1999) suggest that the products of the five PRS genes could aggregate to form subcellular structures which may play a role in maintaining cell integrity. If one assumes that single and multiply deleted strains contain Prs complexes dierent from the wild-type, this could explain the alteration in caeine sensitivity a sreecting a suboptimal maintenance of cell integrity. Human PRPP Synthetase Mammalian Prs isoform subunits are polypeptides of about 35 kDa, with a high degree of homology both within and across species . Purified human PRS subunits, whether recombinant or isolated from cells, undergo concentration-dependent and effector-mediated reversible aggregation in vitro, with enzymatic activity residing in aggregates containing as many as 16 or 32 subunits. Active heteroaggregates of PRS have not been formally demonstrated, but each human recombinant PRS isoform forms active homoaggregates. The human PRS isoform family is composed of 3 polypeptides of identical length. Each human PRS cDNA is encoded by a separate PRPS gene: human PRPS1 and PRPS2 map, respectively, to the long and the short arms of the X chromosome and are widely expressed; PRPS3 maps to human chromosome 7 (Becker 2000) PRPP synthetase exists as complexes consisting of two highly homologous catalytic subunits and other components, termed PRPP synthetase associated proteins (PAPs). The human PAP39 (Phosphoribosyl pyrophosphate synthetase-associated protein shares 44 % sequence identity with the human PRPP synthetase catalytic domain. The presences of PAPs in the PRPP synthetase complexes inhibit the activity of the catalytic subunits. (SGC 2003) It has been shown that mutations in human PRS genes can either activate or inactivate the enzyme, leading to different hereditary disorders including hyperucemia, mental retardation, developmental delay, and other neurological pathologies Overall structure of hPRS1. There are two hPRS1 subunits (coloured in green and yellow respectively) in an asymmetric unit forming a homodimer. Each subunit is composed of two domains related by a pseudo two-fold symmetry and each domain assumes the type I phosphoribosyltransferase fold. The flexible loop involved in ATP binding is shown in blue, the PPi binding loop in magenta, the R5P binding loop in cyan and the flag region in red (for clarity, only one subunit is colour-coded). The bound SO42- ions are shown with ball-and-stick models: Sulf A, the SO42- ions in the R5P binding sites; Sulf B, the SO42- ions in the previously defined allosteric sites; Sulf C, the SO42- ions in the new allosteric sites Image taken from Sheng 2007 Taken from: Arnvig 1990 Regulatory role of PRPP synthetase The essential rate limiting steps in purine biosynthesis occur at the first two steps of the pathway. The synthesis of PRPP by PRPP synthetase is feed-back inhibited by purine-5'-nucleotides (predominantly AMP and GMP). Combinatorial effects of those two nucleotides are greatest, e.g., inhibition is maximal when the correct concentration of both adenine and guanine nucleotides is achieved. The amidotransferase reaction catalyzed by PRPP amidotransferase is also feed-back inhibited allosterically by binding ATP, ADP and AMP at one inhibitory site and GTP, GDP and GMP at another. Conversely the activity of the enzyme is stimulated by PRPP. Inherited Disorders Heredity plays a part in almost all diseases. Recent advances in gene research have allowed a steadily increasing number of specific genes and genetic factors to be linked to a wide variety of medical complaints. There are currently approximately 6,000 known genetic diseases. Those that result from simple mutations of single genes are often referred to as hereditary diseases, and they exhibit distinctive patterns of inheritance in families. Inherited diseases result primarily or exclusively from genetic mutations or genetic imbalance passed on from parent to child at conception. These include Mendelian genetic conditions as well as chromosomal abnormalities. A third group of disorders exists wherein both the environment and genetic factors interact to produce-or influence the course of-a disease. These conditions are often referred to as having multi factorial or complex inheritance patterns. Inherited disorders may be autosomal dominant one copy of the gene is altered by mutation and causes the disease even though the other gene is normal. This is sometimes referred to as vertical transmission because it can be observed in each generation, usually without skipping a generation. Examples of autosomal dominant diseases include achondroplasia (a form of dwarfism), neurofibromatosis, and Huntington disease. PRPP Synthetase related disorders: There are various disorders caused by PRPP Synthetase which can be due to superactivity (gain of function or over expression mutation) or due to reduced activity (loss of function) mutation. Superactivity: Gout: Gout is a disorder that is related to excess production and deposition of uric acid crystals. Uric acid is the byproduct of purine nucleotide catabolism. The root cause of gout is hyperuricemia and it is characterized by recurrent attacks of acute inflammatory arthritis. The formation of urate crystals leads to the formation of tophaceous deposits (sandy, gritty, nodular masses of urate crystals), particularly in the joints which precipitates the episodes of gouty arthritis. Mutations in the PRPS genes that result in superactivity lead to enhanced production of PRPP. Increased levels of PRPP, in turn, drive enhanced de novo synthesis of purine nucleotides in excess of the needs of the body. Thus, the excess purine nucleotides are catabolized resulting in elevated production of uric acid and consequent hyperuricemia and gout. Functional expression studies of all mutations showed that enzyme overactivity was due to alteration of allosteric feedback mechanisms. Arts Syndrome Arts syndrome is an X-linked disorder characterized by mental retardation, early-onset hypotonia, ataxia, delayed motor development, hearing impairment, andopticatrophy. PRPS1 (phosphoribosylpyrophosphatesynthetase1) an isoform in the biochemical step is critical for purine metabolism and nucleotide biosynthesis was studied extensively. The mutations identied were E43D, inpatients with Rosenberg Chutorian syndrome, and M115T, in the Korean patients with CMTX5. Decreased enzyme activity in patients with M115T. PRPS1 is the rst CMT gene that encodes a metabolic enzyme, shedding a new light on the understanding of peripheral nerve specic metabolism and also suggesting the potential of PRPS1 as a target for drugs in prevention and treatment of peripheral neuropathy by antimetabolite therapy. X Linked Deafness In a large 5-generation Chinese family segregating X-linked nonsyndromic hearing loss (NSHL) mapping to the DNF2 locus on chromosome Xq22, analyzed 14 candidate genes and identified a missense mutation in the PRPS1 gene that co segregated with the phenotype. Analysis of the PRPS1 gene in a British American DNF2 family which was previously reported, revealed a different missense mutation; missense mutations were also detected in DFN2 families. It was stated that none of the mutations were predicted to result in a major structural change in the PRPS1 protein, which might explain why the disease phenotype was limited to NSHL. Purine Pyrimidine Pathways and Health Nine genetic disorders have been involved in defects in Pyrimidine metabolism defect and many more in Purine metabolism. Genetic disorders of purine and pyrimidine (PP) metabolism are under-reported and infrequently mentioned in the general literature, as well as in reviews dedicated to other inborn errors of metabolism. Owing to limited awareness, relatively recent recognition, as well as considerable phenotypic variation, these disorders may often be misdiagnosed or remain undiagnosed. Disorders that arise as a result of dysfunction in PP metabolism represent some of the most challenging diagnostic problems in medicine. In addition to their low prevalence rates, they also present with extremely variable signs and symptoms. They may affect any system in a variety of manners, and often mimic other, more recognizable disorders. The diagnostic problem is compounded by the fact that some biochemically affected patients are symptom-free. Rapidly evolving laboratory techniques such as high-performance liquid chromatography coupled to tandem mass spectrometry are now well established as the preferred method for detection for these defects, but currently the most important step in diagnosis consists of suspecting the disorder. Diagnosis is vital because genetic counselling can be provided and, in some cases, specific treatment can be offered that may slow or even reverse clinical symptoms. If undiagnosed, these disorders can be devastating to patients and their families, resulting in early death or institutionalization for the rest of patient's life as succinctly observed by Jurecka in 2009. Thus it is imperative to target these inherent genetic deficiencies which have so long gone unnoticed by development of targeted gene therapies and evolving more sensitive diagnostic technologies. Patients with genetic defects in enzymes crucial to the synthesis or degradation of pyrimidine nucleotides have provided new insights into the vital functions of purines and pyrimidines in peripheral and central nervous system function, muscle function, modulation of blood ow And detoxication reactions , as well as in DNA and RNA synthesis in humans. Such defects underline the important differences between humans and non-mammalian species, and, as such, provide new Avenues for future research. Unique insights into the importance of pyrimidine biosynthesis and metabolism have also resulted particularly their vital role in the normal functioning of the haematological, nervous and, in particular, mitochondrial systems. When defective, the effects can be devastating. It is important to observe that that purine and pyrimidine enzyme defects can also underlie adverse reactions to analogue therapy; for example uridine might be important in male fertility. The pivotal role of UMP, the tissue-and species specicity of particular pathways, and their individual role in healthy humans are underlined by the spectrum of clinical manifestations in all the disorders described. References: Lehninger, A., Nelson, D.L., and Cox, M.M. (2008). Principles of Biochemistry. W. H. Freeman, fifth edition O'Neill. M; (2010). Personal Communication in OMIM (TM) Online Mendelian Inheritance in Man. MIM Number: 311850 Johns Hopkins University, Baltimore, MD. Available at Angstadt, C (1997) "Purine and Pyrimidine Metabolism" Department of Biomedical Sciences. Allegheny University of Health Sciences. Available at < http://library.med.utah.edu/NetBiochem/pupyr/pp.htm> Jimenez, A., Santos, M.A., and Revuelta, J.L. (2008). Phosphoribosyl pyrophosphate synthetase activity affects growth and riboflavin production in ashbya gossypii. BMC Biotechnology, 8:67+. Becker, M. A., M. Kim, et al. (1992). "Regulation of purine nucleotide synthesis in human B lymphoblasts with both hypoxanthine-guanine phosphoribosyltransferase deficiency and phosphoribosylpyrophosphate synthetase superactivity." J Biol Chem 267(7): 4317-4321. Rolfes, R. (2006) "Regulation of purine nucleotide biosynthesis: in yeast and beyond" Biochemical Society Transactions 34, (786-790) Jensen, B (1985) Phosphoribosylpyrophosphate Synthetase of Escherichia coli Properties of the purified enzyme and primary structure of the Prs gene". Journal of Biological Chemistry 15: 6765-6771 Unknown, (2002) "Molecular mechanisms for regulation and catalysis of PRPP synthase" Department of Crystallographic Studies, University of Copenhagen. Available at http://www-ccs.ki.ku.dk/specialer/PRPPsase.htm Schneiter, R (2000) "The importance of the five phosphoribosyl-pyrophosphate synthetase (Prs) gene products of Saccharomyces cerevisiae in the maintenance of cell integrity and the subcellular localization of Prs1p" Microbiology 146, 3269-3278 Carter, A. T., Narbad, A., Pearson, B. M., Beck, K.-F., Logghe, M., Contreras, R. & Schweizer, M. (1994) "Phosphosribosyl pyrophosphate synthetase (PRS) a new gene family in Sacchromyces cerevisae." Yeast (10) 1031-1044 Carter, A. T., Beiche, F., Hove-Jensen, B., Narbad, A., Barker, P. J., Schweizer, L. M. & Schweizer, M. (1997) "PRS1 is a key member of the gene family encoding Phosphoribosyl pyrophosphate synthetase in Saccharomyces cerevisiae". MolGenGenet 254,148156. Becker, M. A. (2001). "Phosphoribosylpyrophosphate synthetase and the regulation of phosphoribosylpyrophosphate production in human cells." Prog Nucleic Acid Res Mol Biol 69: 115-148. de Brouwer, A. P., K. L. Williams, et al. (2007). "Arts syndrome is caused by loss-of-function mutations in PRPS1." Am J Hum Genet 81(3): 507-518. Unknown, (2003) "PRPSAP1: Human phosphoribosylpyrophosphate synthetase-associated protein 1" PDB Code:2C4K SGC Available at < http://www.thesgc.org/structures/structure_description/2C4K/> Sheng, (2007) "Crystal structure of human phosphoribosylpyrophosphate synthetase 1 reveals a novel allosteric site" Biochem. J. 401, 39-47 Jurecka, A (2009). "Inborn errors of purine and pyrimidine metabolism". Journal of Inherited Metabolic Disease 247-263 Lofler, M (2005) "Pyrimidine pathways in health and disease" TRENDS in Molecular Medicine (11)9 421-429 Read More