The sugar transport system discovered by Roseman et al. is commonly referred to as the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS). A parallel pathway in E. coli having to do with the regulation of nitrogen metabolism has been recently identified. The first enzyme of the latter pathway is enzyme INtr (EINtr), which after deletion of the N-terminal 127 amino acids (GAF domain), has 578 amino acids in a sequence homologous to those of other enzymes I. His-356 of EI-Ntr is phosphorylated in the autocatalytic reaction of EI-Ntr with PEP/Mg(II) and this high energy phosphate is transferred to His-16 of NPr, a small carrier protein containing 90 amino acids. Down stream from NPr is enzyme IIA-Ntr, which recently has been implicated in regulating the E. coli K(I) channel transporter TrkA.? ? Interactions between enzyme I-Ntr (EI-Ntr; residues 170-748) and the acceptor protein NPr (residues 1-85) of the nitrogen phosphotansferase system (PTS) have been studied by isothermal titration calorimetry. In the presence of 10 mM sodium phosphate/100 mM NaCl at pH 7.0, the binding stoichiometry is 1:1 with Kd=(5.3 +/- 0.5)mM and deltaH = 2.1 kcal/mol at 293 K, which are approximately the same parameters as observed for binding NPr to the monomeric amino terminal domain of EI-Ntr (EIN-Ntr; residues 170-424 of EI-Ntr). In the latter case, the temperature dependence of deltaH from 10 to 20 degrees C is linear and yields a value for deltaCp of 456 +/- 15 cal K/mol which corresponds to approximately 1600 square Angstrom of apolar surface area burial for EIN-Ntr binding NPr. When the same buffer contained 50 mM sodium phosphate, EI-Ntr has an additional high-affinity site exposed for binding NPr with KD = 0.25 microM and deltaH = 1.4 kcal/mol at 293 K. Values of deltaCp from the temperature dependence of binding enthalpies for the different conditions have been estimated. The dimerization constant for EI-Ntr also is increased 6 to 7-fold at 4 or 20 degrees C by the presence of 50 mM sodium phosphate as determined by sedimentation equilibrium. However, the higher phosphate concentration only decreases the sedimentation coefficient of the EI-Ntr dimer by 0.1 S to S(20,w) = 6.3 S (f/f0 = 1.5). In addition, Trp fluorescence anisotropy measurements show that dissociation of the EINtr dimer is extremely slow as the temperature is shifted from 20 to 4 degrees C. As in the case of the sugar PTS system enzyme I, PEP/Mg(II) binding stabilizes the dimer of EI-Ntr.
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