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.
Highlights
Alexander VinitskyS and Charles Grubmeyersll From the Department of Biology, New York University, New York,New York 10003 and the §Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140
In many cases where ATP hydrolysis drives a chemical reaction, the linkage is mediated by phosphorylated chemical intermediates; for example the reactions catalyzed by glutamine (Midelfort and Rose, 1976) and CTP synthetases
Initialobservations suggested that S. typhimurium NAPRTase catalyzed NAMN synthesis a t rates about the same as ATP hydrolysis when assayed under standard conditions and about 10% of that rate when assayed in the absence of ATP
Summary
A model is presented in which alternation of high and enzyme, nicotinate phosphoribosyltransferase (NAPRTase; low affinity forms of NAPRTase provides a ”steady- EC 2.4.2.11),’ whose coupling of NAMN synthesis and ATP state” coupling between ATP hydrolysis and NAMN hydrolysis provides a novel and important model for energy formation. In many cases where ATP hydrolysis drives a chemical reaction, the linkage is mediated by phosphorylated chemical intermediates; for example the reactions catalyzed by glutamine (Midelfort and Rose, 1976) and CTP synthetases (von der Saal et al, 1985). Such chemically coupled systems are relatively well-explored and understood since coupling is intrinsic to the chemical process.
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