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

Enzyme I of the bacterial phosphoenolpyruvate:sugar phosphotransferase syst em can be phosphorylated by PEP on an active-site histidine residue, locali zed to a cleft between an ex-helical domain and an alpha/beta domain on the amino terminal half of the protein. Tho phosphoryl group on the active-sit e histidine can be passed to an active-site histidine residue of HPr. It ha s been proposed that the major interaction between enzyme I and HPr occurs via the a-helical domain of enzyme I. The isolated recombinant or-helical d omain (residues 25-145) with similar to 80% a-helices as well as enzyme I d eficient in that domain [EI(Delta HD)] with similar to 50% alpha-helix cont ent from RI. capricolum were used to further elucidate the nature of the en zyme I-HPr complex. Isothermal titration calorimetry demonstrated that HPr binds to the a-helical domain and intact enzyme I with K'(A) = 5 x 10(4) an d 1.4 x 10(5) M-1 at pH 7.5 and 25 degrees C, respectively, but not to EI(D elta HD), which contains the active-site histidine of enzyme I and can be a utophosphorylated by PEP, In vitro reconstitution experiments with proteins from both RI. capricolum and E, coli showed that EI(Delta HD) can donate i ts bound phosphoryl group to HPr in the presence of the isolated a-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(Delta HD) pair. Recombinant EIC strongly self-associates (K'(A) approximate to 1 0(10) M-1) in comparison to dimerization constants of 10(5)-10(7) M-1 measu red for EI and EI(Delta HD).