Properties of mutated Rhodospirillum rubrum H+-pyrophosphatase expressed in Escherichia coli.
The membrane-bound proton pumping inorganic pyrophosphate synthase/pyrophosphatase (H(+)-PPi synthase/H(+)-PPase) from the photosynthetic bacterium Rhodospirillum rubrum was functionally expressed in Escherichia coli C43(DE3) cells. Based on a new topology model of the enzyme, charged residues predicted to be located near or within the membrane were selected for site-directed mutagenesis. Several of these mutations resulted in an almost complete inactivation of the enzyme. Four mutated residues appear to show a selective impairment of proton translocation and are thus likely to be involved in coupling pyrophosphate hydrolysis with electrogenic proton pumping. Two of these mutations, R176K and E584D, caused increased tolerance to salt. In addition, the former mutation caused an increased K(m) of one order of magnitude for the hydrolysis reaction. These results and their possible implications for the enzyme function are discussed.
Schultz
Anders
A
Arrhenius Laboratories, Department of Biochemistry and Biophysics, Stockholm University, Svante Arrhenius vag 10-12, S-10691 Stockholm, Sweden.
Baltscheffsky
Margareta
M
D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D043564:Inorganic Pyrophosphatase / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009997:Osmotic Pressure; D017433:Protein Structure, Secondary; D012247:Rhodospirillum rubrum / Q000201:enzymology* / Q000235:genetics
D043564:Inorganic Pyrophosphatase