The sequencing expression, purification, and steady-state kinetic analysis of quinolinate phosphoribosyl transferase from Escherichia coli.
The nadC gene from Escherichia coli was isolated and sequenced. The gene was then cloned into an expression vector and, following transformation, the resulting bacteria were able to produce quinolinate phosphoribosyl transferase as about 2% of the soluble protein. The enzyme was purified in five steps leading to a homogeneous preparation. The enzyme reaction shows an ordered binding mechanism where the magnesium ion complex of 5-phosphoribosyl-1-pyrophosphate binds first followed by quinolinic acid. The products are pyrophosphate CO2, and nicotinate mononucleotide. Product inhibition studies show that nicotinate mononucleotide is a competitive inhibitor with respect to 5-phosphoribosyl-1-pyrophosphate while pyrophosphate is noncompetitive with respect to both 5-phosphoribosyl-1-pyrophosphate and quinolinic acid. Phthalic acid and fructose-1,6-bisphosphate were used as dead-end inhibitors. Phthalate was competitive with respect to quinolinic acid but uncompetitive with respect to 5-phosphoribosyl-1-pyrophosphate. Fructose-1,6-bisphosphate was a competitive inhibitor with respect to 5-phosphoribosyl-1-pyrophosphate and noncompetitive with respect to quinolinic acid. The Km values for the substrates are 15.6 microM for 5-phosphoribosyl-1-pyrophosphate and 6.4 microM for quinolinic acid.
Bhatia
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Department of Chemistry, University of Akron, Ohio 44325-3601, USA.
Calvo
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D000595:Amino Acid Sequence; D001483:Base Sequence; D001667:Binding, Competitive; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D003653:Decarboxylation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D007700:Kinetics; D008969:Molecular Sequence Data; D010430:Pentosyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D010754:Phosphoribosyl Pyrophosphate / Q000378:metabolism; D017378:Quinolinic Acid / Q000378:metabolism; D013379:Substrate Specificity; D000098553:Nicotinate-Nucleotide Diphosphorylase (Carboxylating)
D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D010754:Phosphoribosyl Pyrophosphate; D010430:Pentosyltransferases; D000098553:Nicotinate-Nucleotide Diphosphorylase (Carboxylating); D017378:Quinolinic Acid