title: A new paradigm for biochemical energy coupling. Salmonella typhimurium nicotinate phosphoribosyltransferase.
abstract: The pncB gene of Salmonella typhimurium was used to develop an overexpression system for nicotinate phosphoribosyltransferase (NAPRTase, EC 2.4.2.11), which forms nicotinate mononucleotide (NAMN) and PPi from nicotinate and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). NAPRTase hydrolyzes ATP in 1:1 molar stoichiometry to NAMN synthesis. Hydrolysis of ATP alters the ratio of products/substrates for the reaction nicotinate + PRPP <--> NAMN + PPi from its equilibrium value of 0.67 to a steady-state value of 1100. The energy for the maintenance of this ratio must come from ATP hydrolysis. However, in contrast to other ATP-utilizing enzymes, when all ATP is hydrolyzed the unfavorable product/substrate ratio collapses. ATP/ADP exchange results suggest that the overall reaction involves a phosphoenzyme (E-P) arising from E.ATP. Km values for nicotinate and PRPP each decreased by 200-fold when ATP was present to phosphorylate the enzyme. PPi stimulated the ATPase activity of the enzyme to Vmax values, suggesting that PPi formation during catalysis provides a trigger for cleavage of the putative E-P in the overall reaction and regenerates the low affinity form of the enzyme. A model is presented in which alternation of high and low affinity forms of NAPRTase provides a "steady-state" coupling between ATP hydrolysis and NAMN formation.
authors.affiliation: Department of Biology, New York University, New York 10003.
authors.forename: A
authors.initials: A
authors.lastname: Vinitsky
authors.forename: C
authors.initials: C
authors.lastname: Grubmeyer
mesh_terms: D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D005798:Genes, Bacterial; D007700:Kinetics; D009525:Niacin / Q000378:metabolism; D009537:Nicotinamide Mononucleotide / Q000031:analogs & derivatives / Q000378:metabolism; D010430:Pentosyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010754:Phosphoribosyl Pyrophosphate / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012486:Salmonella typhimurium / Q000201:enzymology* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D009537:Nicotinamide Mononucleotide; D009525:Niacin; C002953:nicotinate mononucleotide; D010754:Phosphoribosyl Pyrophosphate; D010430:Pentosyltransferases; C021864:nicotinate phosphoribosyltransferase