Introduction of a carboxyl group in the first transmembrane helix of Escherichia coli F1Fo ATPase subunit c and cytoplasmic pH regulation.
The multicopy subunit c of the H(+)-transporting F1Fo ATP synthase of Escherichia coli folds across the membrane as a hairpin of two hydrophobic alpha helices. The subunits interact in a front-to-back fashion, forming an oligomeric ring with helix 1 packing in the interior and helix 2 at the periphery. A conserved carboxyl, Asp(61) in E. coli, centered in the second transmembrane helix is essential for H+ transport. A second carboxylic acid in the first transmembrane helix is found at a position equivalent to Ile28 in several bacteria, some the cause of serious infectious disease. This side chain has been predicted to pack proximal to the essential carboxyl in helix 2. It appears that in some of these bacteria the primary function of the enzyme is H+ pumping for cytoplasmic pH regulation. In this study, Ile28 was changed to Asp and Glu. Both mutants were functional. However, unlike the wild type, the mutants showed pH-dependent ATPase-coupled H+ pumping and passive H+ transport through Fo. The results indicate that the presence of a second carboxylate enables regulation of enzyme function in response to cytoplasmic pH and that the ion binding pocket is aqueous accessible. The presence of a single carboxyl at position 28, in mutants I28D/D61G and I28E/D61G, did not support growth on a succinate carbon source. However, I28E/D61G was functional in ATPase-coupled H+ transport. This result indicates that the side chain at position 28 is part of the ion binding pocket.
Jones
P C
PC
Dunn Human Nutrition Unit, Medical Research Council, Cambridge CB2 2XY, United Kingdom. pcj@mrc-dunn.cam.ac.uk
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001224:Aspartic Acid / Q000737:chemistry / Q000235:genetics; D002245:Carbon Dioxide / Q000737:chemistry; D002462:Cell Membrane / Q000737:chemistry / Q000201:enzymology / Q000235:genetics; D003593:Cytoplasm / Q000201:enzymology*; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005973:Glutamine / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D005973:Glutamine; D002245:Carbon Dioxide; C041069:carboxyl radical; D001224:Aspartic Acid; D006180:Proton-Translocating ATPases