Role of a conserved glutamate residue in the Escherichia coli SecA ATPase mechanism.
Escherichia coli SecA uses ATP to drive the transport of proteins across cell membranes. Glutamate 210 in the "DEVD" Walker B motif of the SecA ATP-binding site has been proposed as the catalytic base for ATP hydrolysis (Hunt, J. F., Weinkauf, S., Henry, L., Fak, J. J., McNicholas, P., Oliver, D. B., and Deisenhofer, J. (2002) Science 297, 2018-2026). Consistent with this hypothesis, we find that mutation of glutamate 210 to aspartate results in a 90-fold reduction of the ATP hydrolysis rate compared with wild type SecA, 0.3 s(-1) versus 27 s(-1), respectively. SecA-E210D also releases ADP at a slower rate compared with wild type SecA, suggesting that in addition to serving as the catalytic base, glutamate 210 might aid turnover as well. Our results contradict an earlier report that proposed aspartate 133 as the catalytic base (Sato, K., Mori, H., Yoshida, M., and Mizushima, S. (1996) J. Biol. Chem. 271, 17439-17444). Re-evaluation of the SecA-D133N mutant used in that study confirms its loss of ATPase and membrane translocation activities, but surprisingly, the analogous SecA-D133A mutant retains full activity, revealing that this residue does not play a key role in catalysis.
Zito
Christopher R
CR
Molecular Biology and Biochemistry Department, Wesleyan University, Middletown, Connecticut 06459, USA.
Antony
Edwin
E
Hunt
John F
JF
Oliver
Donald B
DB
Hingorani
Manju M
MM
D000251:Adenosine Triphosphatases / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001412:Bacillus subtilis / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D018698:Glutamic Acid / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D009154:Mutation; D010710:Phosphates / Q000737:chemistry; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D017386:Sequence Homology, Amino Acid; D013997:Time Factors
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D010710:Phosphates; D000069816:SEC Translocation Channels; D001224:Aspartic Acid; D018698:Glutamic Acid; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins; D008274:Magnesium