Enzyme I of the phosphoenolpyruvate : sugar phosphotransferase system. In vitro intragenic complementation: The roles of Arg126 in phosphoryl transfer and the C-terminal domain in dimerization

Enzyme I mutants of the Salmonella typhimurium phosphoenolpyruvute:sugar ph osphotransferase system (PTS), which show in vitro intragenic complementati on, have been identified as Arg126Cys (strain SE 1690 ptsI34), Gly356Ser (s train SB1681 ptsI16), and Arg375Cys (strain SB1476 ptsI17). The mutation Ar g126Cys is in the N-terminal HPr-binding domain, and complements Gly356Ser and Arg375Cys enzyme I mutations located in the C-terminal phosphoenolpyruv ate(PEP)-binding domain. Complementation results in the formation of unstab le heterodimers. None of the mutations alters the K-m for HPr, which is pho sphorylated by enzyme I. Arg126 is a conserved residue; the Arg126Cys mutat ion gives a V-max of 0.04% wild-type, establishing a role in phosphoryl tra nsfer. The Gly356Ser and Arg375Cys mutations reduce enzyme I V-max to 4 and 2%, respectively, and for both, the PEP K-m is increased from 0.1 to 3 mM. It is concluded that this activity was from the monomer, rather than the d imer normally found in assays of wild-type. In the presence of Arg126Cys en zyme, V-max for Gly356Ser and Arg375Cys enzymes I increased 6- and 2-fold, respectively; the K-m for PEP decreased to < 10 mu M, but the K-m became de pendent upon the stability of the heterodimer in the assay. Gly356 is conse rved in enzyme I and pyruvate phosphate dikinase, which is a homologue of e nzyme I, and this residue is part of a conserved sequence in the subunit in teraction site. Gly356Ser mutation impairs enzyme I dimerization. The mutat ion Arg375Cys also impairs dimerization, but the equivalent residue in pyru vate phosphate dikinase is not associated with the subunit interaction site . A 37 000 Da, C-terminal domain of enzyme I has been expressed and purifie d, it dimerizes and complements Gly356Ser and Arg375Cys enzymes I proving t hat the association/dissociation properties of enzyme I are a function of t he C-terminal domain.