Inhibition studies on Rhodospirillum rubrum H(+)-pyrophosphatase expressed in Escherichia coli.
The membrane-bound proton-pumping inorganic pyrophosphatase from Rhodospirillum rubrum was heterologously expressed in Escherichia coli C43(DE3) cells and was inhibited by 4-bromophenacyl bromide (BPB), N,N'-dicyclohexylcarbodiimid (DCCD), diethyl pyrocarbonate (DEPC) and fluorescein 5'-isothiocyanate (FITC). In each case, the enzyme activity was rather well protected against inhibitory action by the substrate Mg(2)PPi. Site-directed mutagenesis was employed in attempts to identify target residues for these inhibitors. D217 and K469 appear to be the prime targets for DCCD and FITC, respectively, and may thus be involved in substrate binding. No major effect on enzyme activities was seen when any one of the four histidine residues present in the enzyme were substituted. Nevertheless, a mutant with all of the four charged histidine residues replaced retained only less than 10% of the hydrolysis and proton-pumping activities. Substitution of D217 with A or H yielded an enzyme with at least an order of magnitude lower hydrolysis activity. In contrast with the wild-type, these variants showed higher hydrolysis rates at lower concentrations of Mg(2+), possibly reflecting a change in substrate preference from Mg(2)PPi to MgPPi. BPB is a H(+)-pyrophosphatase inhibitor that apparently has not been used previously as an inhibitor of these enzymes.
Schultz
Anders
A
Department of Biochemistry and Biophysics, Arrhenius Laboratories, Stockholm University, Svante Arrhenius vag 10-12, S-10691, Stockholm, Sweden.
Baltscheffsky
Margareta
M
D000098:Acetophenones / Q000494:pharmacology; D015153:Blotting, Western; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology; D014644:Genetic Variation; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis / Q000187:drug effects; D043564:Inorganic Pyrophosphatase / Q000037:antagonists & inhibitors* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000494:pharmacology; D011522:Protons; D012247:Rhodospirillum rubrum / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D000098:Acetophenones; D004791:Enzyme Inhibitors; C437127:O,O-diethylphosphorylcholine iodide; D011522:Protons; D010767:Phosphorylcholine; D006639:Histidine; D004024:Dicyclohexylcarbodiimide; D043564:Inorganic Pyrophosphatase; D016650:Fluorescein-5-isothiocyanate; D008274:Magnesium; C006821:4-bromophenacyl bromide