A new paradigm for biochemical energy coupling. Salmonella typhimurium nicotinate phosphoribosyltransferase.
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.
Vinitsky
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A
Department of Biology, New York University, New York 10003.
Grubmeyer
C
C
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
D011994:Recombinant Proteins; D009537:Nicotinamide Mononucleotide; D009525:Niacin; C002953:nicotinate mononucleotide; D010754:Phosphoribosyl Pyrophosphate; D010430:Pentosyltransferases; C021864:nicotinate phosphoribosyltransferase