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  • Title: Interactions at the 2 and 5 positions of 5-phosphoribosyl pyrophosphate are essential in Salmonella typhimurium quinolinate phosphoribosyltransferase.
    Author: Bello Z, Stitt B, Grubmeyer C.
    Journal: Biochemistry; 2010 Feb 23; 49(7):1377-87. PubMed ID: 20047307.
    Abstract:
    Quinolinate phosphoribosyltransferase (QAPRTase, EC 2.4.2.19) catalyzes an unusual phosphoribosyl transfer that is linked to a decarboxylation reaction to form the NAD precursor nicotinate mononucleotide, carbon dioxide, and pyrophosphate from quinolinic acid (QA) and 5-phosphoribosyl 1-pyrophosphate (PRPP). Structural studies and sequence similarities with other PRTases have implicated Glu214, Asp235, Lys153, and Lys284 in contributing to catalysis through direct interaction with PRPP. The four residues were substituted by site-directed mutagenesis. A nadC deletant form of BL21DE3 was created to eliminate trace contamination by chromosomal QAPRTase. The mutant enzymes were readily purified and retained their dimeric aggregation state on gel filtration. Substitution of Lys153 with Ala resulted in an inactive enzyme, indicating its essential nature. Mutation of Glu214 to Ala or Asp caused at least a 4000-fold reduction in k(cat), with 10-fold increases in K(m) and K(D) values for PRPP. However, mutation of Glu214 to Gln had only modest effects on ligand binding and catalysis. pH profiles indicated that the deprotonated form of a residue with pK(a) of 6.9 is essential for catalysis. The WT-like pH profile of the E214Q mutant indicated that Glu214 is not that residue. Mutation of Asp235 to Ala did not affect ligand binding or catalysis. Mutation of Lys284 to Ala decreased k(cat) by 30-fold and increased K(m) and K(D) values for PRPP by 80-fold and at least 20-fold, respectively. The study suggests that Lys153 is necessary for catalysis and important for PRPP binding, Glu214 provides a hydrogen bond necessary for catalysis but does not act as a base or electrostatically to stabilize the transition state, Lys284 is involved in PRPP binding, and Asp235 is not essential.
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