PRPS1 Human
Description
The PRPS1 is fused to 20 a.a His-Tag at N-terminus and purified by proprietary chromatographic techniques.
Product Specs
Product Science Overview
Phosphoribosyl Pyrophosphate Synthetase 1 (PRPS1) is an enzyme that plays a crucial role in the biosynthesis of nucleotides, which are the building blocks of DNA and RNA. This enzyme catalyzes the conversion of ribose 5-phosphate into phosphoribosyl pyrophosphate (PRPP), a key precursor in the synthesis of purine and pyrimidine nucleotides, as well as the cofactors NAD and NADP, and the amino acids histidine and tryptophan . The human recombinant form of PRPS1 is produced through recombinant DNA technology, allowing for its use in various research and therapeutic applications.
The preparation of human recombinant PRPS1 typically involves the following steps:
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Gene Cloning: The PRPS1 gene is amplified from a human cDNA library using polymerase chain reaction (PCR). The primers used for cloning are designed to include restriction sites for subsequent cloning steps .
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Vector Construction: The amplified PRPS1 gene is inserted into an expression vector, such as pET22b(+), using restriction enzymes like NdeI and XhoI. This vector contains elements necessary for the expression of the gene in a host organism .
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Transformation: The recombinant plasmid is introduced into a suitable host organism, commonly Escherichia coli (E. coli) BL21 (DE3), through a process called transformation. This host strain is engineered to express the recombinant protein upon induction .
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Protein Expression: The transformed E. coli cells are cultured, and the expression of PRPS1 is induced using an agent such as isopropyl β-D-1-thiogalactopyranoside (IPTG). The cells are then harvested, and the recombinant protein is extracted .
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Purification: The recombinant PRPS1 protein is purified using techniques such as affinity chromatography, which exploits the specific binding properties of the protein. The purified protein is then analyzed for its activity and purity .
PRPS1 catalyzes the transfer of a diphosphoryl group from ATP to ribose 5-phosphate, resulting in the formation of PRPP and AMP . The reaction mechanism involves the following steps:
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Substrate Binding: Ribose 5-phosphate binds to the active site of PRPS1, followed by the binding of Mg-ATP. The magnesium ion (Mg2+) is essential for stabilizing the ATP molecule .
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Transition State Formation: Upon binding of both substrates, the enzyme undergoes a conformational change, facilitating the transfer of the diphosphate group from ATP to the C1-hydroxy group of ribose 5-phosphate .
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Product Release: The reaction proceeds through an SN2 mechanism, where the anomeric hydroxyl group of ribose 5-phosphate attacks the beta-phosphorus of ATP. This results in the formation of PRPP and the release of AMP .
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Regulation: PRPS1 activity is regulated by feedback inhibition from ADP and GDP, which bind to the enzyme and inhibit its activity. Mutations in PRPS1 can lead to altered enzyme activity and are associated with various metabolic disorders .
