Enzyme I mutants of the Salmonella typhimurium phosphoenolpyruvate:sugar phosphotransferase
system (PTS), which show in vitro intragenic complementation, have been identified
as Arg126Cys (strain SB1690 ptsI34), Gly356Ser (strain SB1681 ptsI16), and Arg375Cys
(strain SB1476 ptsI17). The mutation Arg126Cys is in the N-terminal HPr-binding domain,
and complements Gly356Ser and Arg375Cys enzyme I mutations located in the C-terminal
phosphoenolpyruvate(PEP)-binding domain. Complementation results in the formation
of unstable heterodimers. None of the mutations alters the K(m) for HPr, which is
phosphorylated by enzyme I. Arg126 is a conserved residue; the Arg126Cys mutation
gives a V(max) of 0.04% wild-type, establishing a role in phosphoryl transfer. 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 dimer normally found in assays of wild-type.
In the presence of Arg126Cys enzyme, V(max) for Gly356Ser and Arg375Cys enzymes I
increased 6- and 2-fold, respectively; the K(m) for PEP decreased to <10 microM, but
the K(m) became dependent upon the stability of the heterodimer in the assay. Gly356
is conserved in enzyme I and pyruvate phosphate dikinase, which is a homologue of
enzyme I, and this residue is part of a conserved sequence in the subunit interaction
site. Gly356Ser mutation impairs enzyme I dimerization. The mutation Arg375Cys also
impairs dimerization, but the equivalent residue in pyruvate phosphate dikinase is
not associated with the subunit interaction site. A 37 000 Da, C-terminal domain of
enzyme I has been expressed and purified; it dimerizes and complements Gly356Ser and
Arg375Cys enzymes I proving that the association/dissociation properties of enzyme
I are a function of the C-terminal domain.