tailieunhanh - Báo cáo Y học: Role of conserved residues within helices IV and VIII of the oxaloacetate decarboxylase b subunit in the energy coupling mechanism of the Na+ pump

Mikrobiologisches Institut der Eidgeno¨ssischen Technischen Hochschule, ETH-Zentrum, Zu¨rich, Switzerland The membrane-bound b subunit of the oxaloacetate decarboxylase Na+ pump of Klebsiella pneumoniae catalyzes the decarboxylation of enzyme-bound biotin. This event is coupled to the transport of 2 Na+ ions into the periplasm and consumes a periplasmically derived proton. The connecting fragment IIIa and transmembrane helices IV and VIII of the b subunit are highly conserved, harboring residues D203, Y229, N373, G377, S382, and R389 that play a profound role in catalysis. . | Eur. J. Biochem. 269 2997-3004 2002 FEBS 2002 doi Role of conserved residues within helices IV and VIII of the oxaloacetate decarboxylase b subunit in the energy coupling mechanism of the Na pump Markus Schmid Thomas Vorburger Klaas Martinus Pos and Peter Dimroth Mikrobiologisches Institut der Eidgenossischen Technischen Hochschule ETH-Zentrum Zurich Switzerland The membrane-bound b subunit of the oxaloacetate decarboxylase Na pump of Klebsiella pneumoniae catalyzes the decarboxylation of enzyme-bound biotin. This event is coupled to the transport of 2 Na ions into the periplasm and consumes a periplasmically derived proton. The connecting fragment IIIa and transmembrane helices IV and VIII of the b subunit are highly conserved harboring residues D203 Y229 N373 G377 S382 and R389 that play a profound role in catalysis. We report here detailed kinetic analyses of the wild-type enzyme and the b subunit mutants N373D N373L S382A S382D S382t R389A and R389D. In these studies pH profiles Na binding affinities Hill coefficients Vmax values and inhibition by Na was determined. A prominent result is the complete lack of oxalo-acetate decarboxylase activity of the S382A mutant at Na concentrations up to 20 mM and recovery of significant activities at elevated Na concentrations KNa w 400 mMat pH where the wild-type enzyme is almost completely inhibited. These results indicate impaired Na binding to the S382 including site in the S382A mutant. Oxaloacetate decarboxylation by the S382A mutant at high Na concentrations is uncoupled from the vectorial events of Na or H translocation across the membrane. Based on all data with the mutant enzymes we propose a coupling mechanism which includes Na binding to center I contributed by D203 region IIIa and N373 helix VIII and center II contributed by Y229 helix IV and S382 helix VIII . These centers are exposed to the cytoplasmic surface in the carboxybiotinbound state of the b subunit and become .

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