Identifying groups involved in the binding of prephenate to prephenate dehydrogenase from Escherichia coli

Site-directed mutagenesis was used to investigate the importance of Lys178, Arg286, and Arg294 in the binding of prephenate to the bifunctional enzyme chorismate mutase-prephenate dehydrogenase, From comparison of the kinetic parameters of wild-type enzyme and selected mutants, we conclude that only Arg294 interacts specifically with prephenate. The R294Q substitution redu ces the enzyme's affinity for prephenate without affecting V/E-t of the deh ydrogenase reaction or the kinetic parameters of the mutase reaction. Arg29 4 likely interacts with the ring carboxylate at C-l of prephenate since the dissociation constants for a series of inhibitors missing the ring carboxy l group were similar for wild-type and R294Q enzymes. The pH dependencies o f log (V/KprephenateEt) and of pK(i) for hydroxyphenyllactate show that the wild-type dehydrogenase possesses a group with a pK of 8.8 that must be pr otonated for binding prephenate to the enzyme. None of the three conserved residues is this group since its titration is observed in the V/Kprephenate Et profiles for the mutants K178Q, R286A, and R294Q. This group is also see n in the pH-rate profiles of the binding of two substrate analogues, hydrox yphenyllactate and deoxoprephenate. Their only common structural feature at C-l is the side chain carboxylate, indicating that the protonated residue (pK 8.8) must interact with prephenate's side chain carboxylate. Gdn-HCl-in duced denaturation was conducted on wild-type and selected mutant proteins. Unfolding of the wild-type enzyme proceeds through a partially unfolded di mer which dissociates into unfolded monomers. The order of stability is wil d-type = R294Q > K178Q > R286A > K178R. The least unstable mutants have red uced mutase and dehydrogenase activities.