TY - JOUR
T1 - Specific placement of tryptophan in the catalytic sites of Escherichia coli F1-ATPase provides a direct probe of nucleotide binding
T2 - Maximal ATP hydrolysis occurs with three sites occupied
AU - Weber, Joachim
AU - Wilke-Mounts, Susan
AU - Lee, Rita S.F.
AU - Grell, Ernst
AU - Senior, Alan E.
PY - 1993/9/25
Y1 - 1993/9/25
N2 - Residue βY331 of Escherichia coli F1-ATPase is known from previous affinity labeling, mutagenesis, and linbenzo-ADP binding experiments to interact directly with the adenine moiety of substrates bound in catalytic sites. Here we mutagenized βY331 to tryptophan. Mutant cells grew well on succinate or limiting glucose; purified mutant F1 had kcat/Km and lin-benzo-ADP binding characteristics similar to wild type. Fluorescence from βW331 residues exhibited a maximum at 349 nm, indicating a polar environment in unoccupied sites. ATP, ADP, or AMPPNP caused virtually complete quenching of βW331 fluorescence, so that the fluorescence of mutant F1 with occupied catalytic sites resembled that of wild-type enzyme. Therefore the βW331 fluorescence provided a direct probe of nucleotide binding to catalytic sites under true equilibrium conditions. We measured ATP binding and hydrolysis in parallel experiments and found that occupancy of one or two catalytic sites per F1 molecule did not yield significant rates of hydrolysis while occupancy of all three sites yielded Vmax rates. Km(ATP) was similar to Kd3, the Kd for ATP binding to the third catalytic site. We also measured AMPPNP and ADP binding parameters. For ADP, the "on" rate at the first catalytic site was much faster (≥5 × 105 M-1 s-1) than seen previously by centrifuge column procedures, although the Kd was not much changed. For AMPPNP, the "on" rate at the first site was 2 orders of magnitude less than for ADP or ATP, and the Kd was similar to that for ADP.
AB - Residue βY331 of Escherichia coli F1-ATPase is known from previous affinity labeling, mutagenesis, and linbenzo-ADP binding experiments to interact directly with the adenine moiety of substrates bound in catalytic sites. Here we mutagenized βY331 to tryptophan. Mutant cells grew well on succinate or limiting glucose; purified mutant F1 had kcat/Km and lin-benzo-ADP binding characteristics similar to wild type. Fluorescence from βW331 residues exhibited a maximum at 349 nm, indicating a polar environment in unoccupied sites. ATP, ADP, or AMPPNP caused virtually complete quenching of βW331 fluorescence, so that the fluorescence of mutant F1 with occupied catalytic sites resembled that of wild-type enzyme. Therefore the βW331 fluorescence provided a direct probe of nucleotide binding to catalytic sites under true equilibrium conditions. We measured ATP binding and hydrolysis in parallel experiments and found that occupancy of one or two catalytic sites per F1 molecule did not yield significant rates of hydrolysis while occupancy of all three sites yielded Vmax rates. Km(ATP) was similar to Kd3, the Kd for ATP binding to the third catalytic site. We also measured AMPPNP and ADP binding parameters. For ADP, the "on" rate at the first catalytic site was much faster (≥5 × 105 M-1 s-1) than seen previously by centrifuge column procedures, although the Kd was not much changed. For AMPPNP, the "on" rate at the first site was 2 orders of magnitude less than for ADP or ATP, and the Kd was similar to that for ADP.
UR - http://www.scopus.com/inward/record.url?scp=0027317040&partnerID=8YFLogxK
M3 - Article
C2 - 8376371
AN - SCOPUS:0027317040
SN - 0021-9258
VL - 268
SP - 20126
EP - 20133
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 27
ER -