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 Klebsiella pneumoniae catalyzes the decarboxylation of carboxybiotin, which is coupled to Na(+) translocation and consumes a periplasmically derived proton. Upon site-directed mutagenesis of 20 polar and/or conserved residues within putative membrane-integral regions, the specific oxaloacetate decarboxylase activities were reduced to various extents, but only the enzyme with a Y229F mutation was completely inactive. We propose that Y229 is part of the network 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(+) binding, because in the Y229F orY229A mutants, the beta-subunit was protected from tryptic digestion by 50 mM NaCl like in the wild-type enzyme. Oxaloacetate decarboxylase with a betaC291E mutation was unstable in the absence of Na(+) and dissociated into an alpha-gamma subcomplex and the beta-subunit. The enzyme could only be isolated in the presence of 0. 5 M NaCl. These results are consistent with the notion that the beta-subunit changes its conformation upon Na(+) binding.
Jockel
P
P
Institut für Mikrobiologie der Eidgenössischen Technischen Hochschule, ETH-Zentrum, CH-8092 Zürich, Switzerland.
Schmid
M
M
Choinowski
T
T
Dimroth
P
P
D019943:Amino Acid Substitution / Q000235:genetics; D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D002262:Carboxy-Lyases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D017124:Conserved Sequence / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000201:enzymology*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D062907:Oxaloacetic Acid / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
D008565:Membrane Proteins; D011994:Recombinant Proteins; D062907:Oxaloacetic Acid; D014443:Tyrosine; C024582:N1'-carboxybiotin; D001710:Biotin; D012964:Sodium; D014357:Trypsin; D002262:Carboxy-Lyases; C021916:oxaloacetate decarboxylase; D000254:Sodium-Potassium-Exchanging ATPase