title: Subunit gamma of the oxaloacetate decarboxylase Na(+) pump: interaction with other subunits/domains of the complex and binding site for the Zn(2+) metal ion.
abstract: The oxaloacetate decarboxylase Na(+) pump of Klebsiella pneumoniae is an enzyme complex composed of the peripheral alpha subunit and the two integral membrane-bound subunits beta and gamma. The alpha subunit consists of the N-terminal carboxyltransferase domain and the C-terminal biotin domain, which are connected by a flexible proline/alanine-rich linker peptide. To probe interactions between the two domains of the alpha subunit and between alpha-subunit domains and the gamma subunit, the relevant polypeptides were synthesized in Escherichia coli and subjected to copurification studies. The two alpha-subunit domains had no distinct affinity toward each other and could, therefore, not be purified as a unit on avidin-sepharose. The two domains reacted together catalytically, however, performing the carboxyl transfer from oxaloacetate to protein-bound biotin. This reaction was enhanced up to 6-fold in the presence of the Zn(2+)-containing gamma subunit. On the basis of copurification with different tagged proteins, the C-terminal biotin domain but not the N-terminal carboxyltransferase domain of the alpha subunit formed a strong complex with the gamma subunit. Upon the mutation of gamma H78 to alanine, the binding affinity to subunit alpha was lost, indicating that this amino acid may be essential for formation of the oxaloacetate decarboxylase enzyme complex. The binding residues for the Zn(2+) metal ion were identified by site-directed and deletion mutagenesis. In the gamma D62A or gamma H77A mutant, the Zn(2+) content of the decarboxylase decreased to 35% or 10% of the wild-type enzyme, respectively. Less than 5% of the Zn(2+) present in the wild-type enzyme was found if the two C-terminal gamma-subunit residues H82 and P83 were deleted. Corresponding with the reduced Zn(2+) contents in these mutants, the oxaloacetate decarboxylase activities were diminished. These results indicate that aspartate 62, histidine 77, and histidine 82 of the gamma subunit are ligands for the catalytically important Zn(2+) metal ion.
authors.affiliation: Mikrobiologisches Institut der Eidgenössischen Technischen Hochschule, ETH-Zentrum, CH-8092 Zürich, Switzerland.
authors.forename: Markus
authors.initials: M
authors.lastname: Schmid
authors.forename: Markus R
authors.initials: MR
authors.lastname: Wild
authors.forename: Pius
authors.initials: P
authors.lastname: Dahinden
authors.forename: Peter
authors.initials: P
authors.lastname: Dimroth
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D007711:Klebsiella pneumoniae / Q000201:enzymology; D016296:Mutagenesis; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D002262:Carboxy-Lyases; C021916:oxaloacetate decarboxylase; D000254:Sodium-Potassium-Exchanging ATPase; D015032:Zinc