Subunit γ of the Oxaloacetate Decarboxylase Na+ Pump:  Interaction with Other Subunits/Domains of the Complex and Binding Site for the Zn2+ Metal Ion

• Published in: Biochemistry (Easton) 2002-01, Vol.41 (4), p.1285-1292
• Main Authors: Schmid, Markus, Wild, Markus R, Dahinden, Pius, Dimroth, Peter
• Format: Article
• Language: English
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi015764l

The oxaloacetate decarboxylase Na+ pump of Klebsiella pneumoniae is an enzyme complex composed of the peripheral α subunit and the two integral membrane-bound subunits β and γ. The α 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 α subunit and between α-subunit domains and the γ subunit, the relevant polypeptides were synthesized in Escherichia coli and subjected to copurification studies. The two α-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 Zn2+-containing γ subunit. On the basis of copurification with different tagged proteins, the C-terminal biotin domain but not the N-terminal carboxyltransferase domain of the α subunit formed a strong complex with the γ subunit. Upon the mutation of γH78 to alanine, the binding affinity to subunit α was lost, indicating that this amino acid may be essential for formation of the oxaloacetate decarboxylase enzyme complex. The binding residues for the Zn2+ metal ion were identified by site-directed and deletion mutagenesis. In the γD62A or γH77A mutant, the Zn2+ content of the decarboxylase decreased to 35% or 10% of the wild-type enzyme, respectively. Less than 5% of the Zn2+ present in the wild-type enzyme was found if the two C-terminal γ-subunit residues H82 and P83 were deleted. Corresponding with the reduced Zn2+ contents in these mutants, the oxaloacetate decarboxylase activities were diminished. These results indicate that aspartate 62, histidine 77, and histidine 82 of the γ subunit are ligands for the catalytically important Zn2+ metal ion.