Essential role of tyrosine 229 of the oxaloacetate decarboxylase beta-subunit in the energy coupling mechanism of the Na+ pump

The membrane-bound beta-subunit of oxaloacetate decarboxylase from Klebsiel la pneumoniae catalyzes the decarboxylation of carboxybiotin, which is coup led to Na+ translocation and consumes a periplasmically derived proton. Upo n site-directed mutagenesis of 20 polar and/or conserved residues within pu tative membrane-integral regions, the specific oxaloacetate decarboxylase a ctivities were reduced to various extents, but only the enzyme with a Y229F mutation was completely inactive. We propose that Y229 is part of the netw ork by which the proton of S382 is delivered to carboxybiotin, where it is consumed upon catalyzing the immediate decarboxylation of this acid-labile compound. Unlike S382 or D203, Y229 appears to be not involved in Na+ bindi ng, because in the Y229F orY229A mutants, the beta-subunit was protected fr om tryptic digestion by 50 mM NaCl like in the wild-type enzyme. Oxaloaceta te decarboxylase with a beta C291E mutation was unstable in the absence of Na+ and dissociated into an alpha-gamma subcomplex and the beta-subunit. Th e enzyme could only be isolated in the presence of 0.5 M NaCl. These result s are consistent with the notion that the beta-subunit changes its conforma tion upon Na+ binding.