Coexisting kinetically distinguishable forms of dialkylglycine decarboxylase engendered by alkali metal ions.

The pyridoxal phosphate (PLP) dependent enzyme dialkylglycine decarboxylase (DGD) specifically binds alkali metal ions near the active site. Large ions (Rb+, K+) activate the enzyme while smaller ones (Na+, Li+) inhibit it. Crystallographic results have shown that DGD undergoes a metal ion size dependent structural switch [Hohenester, E., Keller, J. W., and Jansonius, J. N. (1994) Biochemistry 33, 13561], but no evidence for multiple conformations in crystalline DGD was obtained. Here, evidence is presented that DGD-K+ in solution exists in two conformations differing in catalytic competence. Initial rate traces for DGD-K+ exhibit a high degree of curvature due to decreasing activity over time. DGD remains tetrameric under the assay conditions as demonstrated by gel filtration experiments, arguing against the possibility of subunit dissociation as the source of activity loss. Likewise, the mass spectrum of DGD shows a single covalent form. A hysteretic model that assumes two slowly interconverting enzyme forms accounts well for the initial rate data when kinetic parameters from biphasic pre-steady-state kinetics are employed. The fit of the model to the data yields an estimate of 59 +/- 1% for the fast form. A cooperative model cannot account for the data. Double reciprocal plots for coenzyme binding to DGD exhibit two linear phases. Similarly, two kinetic phases are observed in PLP association kinetics. The substitution of Na+ or Rb+ for K+ alters the steady-state kinetic parameters of DGD. Preincubation of DGD-K+ with the competitive inhibitor 1-aminocyclopropane-1-carboxylate (ACC) lowers both kcat and KAIB apparently by drawing the enzyme toward the less active, tighter binding form observed in the pre-steady-state kinetics. These results suggest that the structure of the protein around the alkali metal ion determines the conformational distribution. The transamination reaction with l-alanine was coupled in the pre-steady-state to the LDH-catalyzed oxidation of NADH. This experiment yields an estimate of 68 +/- 4% for the fast form, in agreement with the hysteretic fit to the steady-state data. The reaction of DGD with dithiobis(nitrobenzoate) was used to probe the preexisting forms of DGD. Preincubation of DGD with ACC, like the exchange of Na+ for K+, shifts the conformational distribution, in agreement with the steady-state kinetics. These experiments clearly demonstrate that DGD is a hysteretic enzyme whose conformational distribution is controlled by the identity of the alkali metal ion bound near the active site, and that cooperativity does not play a role in catalysis or regulation.