Histidine residues are involved in translocation-coupled ATP hydrolysis by the Sec-A protein.
Treatment of SecA, an essential component of the protein translocation machinery of Escherichia coli, with a histidine-specific reagent, diethylpyrocarbonate, caused significant abolition of its translocation-coupled ATPase (translocation ATPase) activity, which requires a presecretory protein and membrane vesicles, whereas its endogenous ATPase (SecA-ATPase) activity was enhanced by a factor of 2. Diethylpyrocarbonate-treated SecA exhibited an absorption maximum at 240 nm due to the formation of N-carbethoxyhistidine. Upon the modification of about 5 of the total 22 histidine residues in the SecA molecule, both the abolition of its translocation ATPase activity and the enhancement of its SecA-ATPase activity occurred. Intact and modified SecA exhibited similar affinities for ATP, proOmpA and membranes, whereas Vmax of the translocation ATPase activity was significantly lower in the case of the modified SecA. ATP had no effect on the modification of SecA. Taken together, these results indicate that histidine residues susceptible to diethylpyrocarbonate are essential for the translocation ATPase, but not directly involved in the binding of ATP, proOmpA and membranes. A possible reason for the abolition of translocation ATPase is discussed.
Tokuda
H
H
Institute of Molecular and Cellular Biosciences, University of Tokyo, Japan.
Yamanaka
M
M
Mizushima
S
S
D000251:Adenosine Triphosphatases / Q000187:drug effects / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001693:Biological Transport, Active / Q000187:drug effects; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins; D006639:Histidine / Q000378:metabolism*; D006868:Hydrolysis; D026901:Membrane Transport Proteins; D011498:Protein Precursors / Q000378:metabolism; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D011498:Protein Precursors; D000069816:SEC Translocation Channels; C065493:SecE protein, E coli; C057841:SecY protein, E coli; D006639:Histidine; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins; D004047:Diethyl Pyrocarbonate