H+-pyrophosphatase of Rhodospirillum rubrum. High yield expression in Escherichia coli and identification of the Cys residues responsible for inactivation my mersalyl.
H(+)-translocating pyrophosphatase (H(+)-PPase) of the photosynthetic bacterium Rhodospirillum rubrum was expressed in Escherichia coli C43(DE3) cells. Recombinant H(+)-PPase was observed in inner membrane vesicles, where it catalyzed both PP(i) hydrolysis coupled with H(+) transport into the vesicles and PP(i) synthesis. The hydrolytic activity of H(+)-PPase in E. coli vesicles was eight times greater than that in R. rubrum chromatophores but exhibited similar sensitivity to the H(+)-PPase inhibitor, aminomethylenediphosphonate, and insensitivity to the soluble PPase inhibitor, fluoride. Using this expression system, we showed that substitution of Cys(185), Cys(222), or Cys(573) with aliphatic residues had no effect on the activity of H(+)-PPase but decreased its sensitivity to the sulfhydryl modifying reagent, mersalyl. H(+)-PPase lacking all three Cys residues was completely resistant to the effects of mersalyl. Mg(2+) and MgPP(i) protected Cys(185) and Cys(573) from modification by this agent but not Cys(222). Phylogenetic analyses of 23 nonredundant H(+)-PPase sequences led to classification into two subfamilies. One subfamily invariably contains Cys(222) and includes all known K(+)-independent H(+)-PPases, whereas the other incorporates a conserved Cys(573) but lacks Cys(222) and includes all known K(+)-dependent H(+)-PPases. These data suggest a specific link between the incidence of Cys at positions 222 and 573 and the K(+) dependence of H(+)-PPase.
Belogurov
Georgiy A
GA
Department of Biochemistry and Food Chemistry, University of Turku, FIN-20014 Turku, Finland.
Turkina
Maria V
MV
Penttinen
Anni
A
Huopalahti
Saila
S
Baykov
Alexander A
AA
Lahti
Reijo
R
D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D006859:Hydrogen / Q000378:metabolism; D006868:Hydrolysis; D043564:Inorganic Pyrophosphatase; D008634:Mersalyl / Q000494:pharmacology*; D010802:Phylogeny; D010957:Plasmids / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D021381:Protein Transport; D011755:Pyrophosphatases / Q000096:biosynthesis* / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D013997:Time Factors
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D008634:Mersalyl; D006859:Hydrogen; D011755:Pyrophosphatases; D043564:Inorganic Pyrophosphatase; D003545:Cysteine; D011188:Potassium