ASPARTATE-203 OF THE OXALOACETATE DECARBOXYLASE BETA-SUBUNIT CATALYZES BOTH THE CHEMICAL AND VECTORIAL REACTION OF THE NA+ PUMP

We report here a new mode of coupling between the chemical and vectori al reaction explored for the oxaloacetate decarboxylase Na+ pump from Klebsiella pneumoniae. The membrane-bound beta-subunit is responsible for the decarboxylation of carboxybiotin and the coupled translocation of Na+ ions across the membrane. The biotin prosthetic group which is attached to the alpha-subunit becomes carboxylated by carboxyltransfe r from oxaloacetate. The two conserved aspartic acid residues within p utative membrane-spanning domains of the beta-subunit (Asp149 and Asp2 03) were exchanged by site-directed mutagenesis. Mutants D149Q and D14 9E retained oxaloacetate decarboxylase and Na+ transport activities. M utants D203N and D203E, however, had lost these two activities, but re tained the ability to form the carboxybiotin enzyme. Direct participat ion of Asp203 in the catalysis of the decarboxylation reaction is ther efore indicated. In addition, all previous and present data on the enz yme support a model in which the same aspartic acid residue provides a binding site for the metal ion catalysing its movement across the mem brane. The model predicts that Asp203 in its dissociated form binds Na + and promotes its translocation, while the protonated residue transfe rs the proton to the acidlabile carboxybiotin which initiates its deca rboxylation. Strong support for the model comes from the observation t hat Na+ transport by oxaloacetate decarboxylation is accompanied by H transport in the opposite direction. The inhibition of oxaloacetate d ecarboxylation by high Na+ concentrations in a pH-dependent manner is also in agreement with the model.