Ribose-phosphate diphosphokinase transfers the diphosphoryl group from Mg-ATP to ribose 5-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.
Structure
Crystallization and X-ray diffraction studies elucidated the structure of the enzyme, which was isolated by cloning, protein expression, and purification techniques. One subunit of ribose-phosphate diphosphokinase consists of 318 amino acids; the active enzyme complex consists of three homodimers. The structure of one subunit is a five-stranded parallel beta sheet surrounded by four alpha helices at the N-terminal domain and five alpha helices at the C-terminal domain, with two short anti-parallel beta-sheets extending from the core. The catalytic site of the enzyme binds ATP and ribose 5-phosphate. The flexible loop, pyrophosphate binding loop, and flag region comprise the ATP binding site, located at the interface between two domains of one subunit. The flexible loop is so named because of its large variability in conformation. The ribose 5-phosphate binding site consists of residues Asp220–Thr228, located in the C-terminal domain of one subunit. The allosteric site, which binds ADP, consists of amino acid residues from three subunits.
Function
The product of this reaction, phosphoribosyl pyrophosphate, is used in numerous biosynthesis pathways. PRPP provides the ribose sugar in de novo synthesis of purines and pyrimidines, used in the nucleotide bases that form RNA and DNA. PRPP reacts with orotate to form orotidylate, which can be converted to uridylate. UMP can then be converted to the nucleotidecytidine triphosphate. The reaction of PRPP, glutamine, and ammonia forms 5-Phosphoribosyl-1-amine, a precursor to inosinate, which can ultimately be converted to adenosine triphosphate or guanosine triphosphate. PRPP plays a role in purinesalvage 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.
Regulation
Ribose-phosphate diphosphokinase requires Mg2+ for activity; the enzyme acts only on ATP coordinated with Mg2+. 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. However, at high concentrations, phosphate is shown to have an inhibitory effect by competing with the substrate ribose 5-phosphate for binding at the active site. 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.
Role in disease
Because its product is a key compound in many biosynthetic pathways, ribose-phosphate diphosphokinase is involved in some rare disorders and X-linked recessive diseases. Mutations that lead to super-activity result in purine and uric acid overproduction. Super-activity symptoms include gout, sensorineural hearing loss, weak muscle tone, impaired muscle coordination, hereditary peripheral neuropathy, and neurodevelopmental disorder. Mutations that lead to loss-of-function in ribose-phosphate diphosphokinase result in Charcot-Marie-Tooth disease and ARTS syndrome.
