Reconstitution studies using the helical and carboxy-terminal domains of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system.

Abstract

Enzyme I of the bacterial phosphoenolpyruvate:sugar phosphotransferase system can be phosphorylated by PEP on an active-site histidine residue, localized to a cleft between an alpha-helical domain and an alpha/beta domain on the amino terminal half of the protein. The phosphoryl group on the active-site histidine can be passed to an active-site histidine residue of HPr. It has been proposed that the major interaction between enzyme I and HPr occurs via the alpha-helical domain of enzyme I. The isolated recombinant alpha-helical domain (residues 25-145) with approximately 80% alpha-helices as well as enzyme I deficient in that domain [EI(DeltaHD)] with approximately 50% alpha-helix content from M. capricolum were used to further elucidate the nature of the enzyme I-HPr complex. Isothermal titration calorimetry demonstrated that HPr binds to the alpha-helical domain and intact enzyme I with = 5 x 10(4) and 1.4 x 10(5) M(-)(1) at pH 7.5 and 25 degrees C, respectively, but not to EI(DeltaHD), which contains the active-site histidine of enzyme I and can be autophosphorylated by PEP. In vitro reconstitution experiments with proteins from both M. capricolum and E. coli showed that EI(DeltaHD) can donate its bound phosphoryl group to HPr in the presence of the isolated alpha-helical domain. Furthermore, M. capricolum recombinant C-terminal domain of enzyme I (EIC) was shown to reconstitute phosphotransfer activity with recombinant N-terminal domain (EIN) approximately 5% as efficiently as the HD-EI(DeltaHD) pair. Recombinant EIC strongly self-associates ( approximately 10(10) M(-)(1)) in comparison to dimerization constants of 10(5)-10(7) M(-)(1) measured for EI and EI(DeltaHD).