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 reduces the enzyme's affinity for prephenate without affecting V/Et of the dehydrogenase reaction or the kinetic parameters of the mutase reaction. Arg294 likely interacts with the ring carboxylate at C-1 of prephenate since the dissociation constants for a series of inhibitors missing the ring carboxyl group were similar for wild-type and R294Q enzymes. The pH dependencies of log (V/KprephenateEt) and of pKi for hydroxyphenyllactate show that the wild-type dehydrogenase possesses a group with a pK of 8.8 that must be protonated for binding prephenate to the enzyme. None of the three conserved residues is this group since its titration is observed in the V/KprephenateEt profiles for the mutants K178Q, R286A, and R294Q. This group is also seen in the pH-rate profiles of the binding of two substrate analogues, hydroxyphenyllactate and deoxoprephenate. Their only common structural feature at C-1 is the side chain carboxylate, indicating that the protonated residue (pK 8.8) must interact with prephenate's side chain carboxylate. Gdn-HCl-induced denaturation was conducted on wild-type and selected mutant proteins. Unfolding of the wild-type enzyme proceeds through a partially unfolded dimer which dissociates into unfolded monomers. The order of stability is wild-type = R294Q > K178Q > R286A > K178R. The least unstable mutants have reduced mutase and dehydrogenase activities.