COEXISTING KINETICALLY DISTINGUISHABLE FORMS OF DIALKYLGLYCINE DECARBOXYLASE ENGENDERED BY ALKALI-METAL IONS

The pyridoxal phosphate (PLP) dependent enzyme dialkylglycine decarbox ylase (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 evid ence for multiple conformations in crystalline DGD was obtained. Here, evidence is presented that DGD-K+ in solution exists in two conformat ions differing in catalytic competence. Initial rate traces for DGD-K exhibit a high degree of curvature due to decreasing activity over ti me. DGD remains tetrameric under the assay conditions as demonstrated by gel filtration experiments, arguing against the possibility of subu nit dissociation as the source of activity loss. Likewise, the mass sp ectrum of DGD shows a single covalent form. A hysteretic model that as sumes two slowly interconverting enzyme forms accounts well for the in itial rate data when kinetic parameters from biphasic pre-steady-state kinetics are employed. The fit of the model to the data yields an est imate of 59 +/- 1% for the fast form. A cooperative model cannot accou nt for the data. Double reciprocal plots for coenzyme binding to DGD e xhibit two linear phases. Similarly, two kinetic phases are observed i n PLP association kinetics. The substitution of Na+ or Rb+ for K+ alte rs the steady-state kinetic parameters of DGD. Preincubation of DGD-K with the competitive inhibitor 1-aminocyclopropane-1-carboxylate (ACC ) lowers both k(cat) and K-AlB apparently by drawing the enzyme toward the less active, tighter binding form observed in the presteady-state kinetics, These results suggest that the structure of the protein aro und the alkali metal ion determines the conformational distribution. T he transamination reaction with L-alanine was coupled in the pre-stead y-state to the LDH-catalyzed oxidation of NADH. This experiment yields an estimate of 68 +/- 4% for the fast form, in agreement with the hys teretic fit to the steady-state data. The reaction of DGD with dithiob is(nitrobenzoate) was used to probe the preexisting forms of DGD, Prei ncubation of DGD with ACC, like the exchange of Na+ for K+, shifts the conformational distribution, in agreement with the steady-state kinet ics. These experiments clearly demonstrate that DGD is a hysteretic en zyme whose conformational distribution is controlled by the identity o f the alkali metal ion bound near the active site, and that cooperativ ity does not play a role in catalysis or regulation.