Probing the specificity of nucleotide binding to the F1-ATPase from thermophilic Bacillus PS3 and its isolated alpha and beta subunits with 2-N3-[beta, gamma-32P]ATP.
Irradiation of the F1-ATPase from Bacillus PS3 (TF1) in the presence of 134 microM 2-N3-[beta, gamma-32P]ATP plus Mg2+ for 90 min led to 95% inactivation of the ATPase activity which was accompanied by exclusive labeling of the beta subunit. The isolated alpha and beta subunits were also treated separately with 2-N3-[beta, gamma-32P]ATP under similar conditions. Fractionation of a tryptic digest of photolabeled TF1 by reversed-phase HPLC resolved a major and a minor radioactive peptide. Sequence analyses showed that the major peptide contained labeled Tyr-beta 364, whereas the minor one contained labeled Tyr-beta 341, residues known to be part of noncatalytic and catalytic sites, respectively. Two closely eluting radioactive peptides were obtained when a tryptic digest of the photolabeled, isolated beta subunit was fractionated by HPLC. Sequence analyses revealed that both contained labeled Tyr-beta 341. Fractionation of a tryptic digest of the photolabeled, isolated alpha subunit by HPLC resolved two peptides which contained the majority of the radioactivity incorporated. When subjected to eight cycles of automatic Edman degradation, one gave the sequence APGVXDR, corresponding to residues 133-139, in which X is a gap and corresponds to Met-alpha 137, which presumably is the derivatized residue. Only the first five cycles yielded phenylthiohydantoin derivatives when the other radioactive peptide derived from the alpha subunit was submitted to automatic Edman degradation which revealed the sequence APGVM, suggesting that Asp-alpha 138 is derivatized. The overall results suggest that the isolated beta subunit is a useful model for studying binding of nucleotides to catalytic sites, whereas the isolated alpha subunit may be of limited value in modeling interactions of nucleotides with noncatalytic sites.
Jault
J M
JM
Department of Chemistry, University of California at San Diego, La Jolla 92093-0601.
Kaibara
C
C
Yoshida
M
M
Garrod
S
S
Allison
W S
WS
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D001386:Azides / Q000378:metabolism*; D001407:Bacillus / Q000201:enzymology*; D001665:Binding Sites; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry; D006180:Proton-Translocating ATPases / Q000378:metabolism* / Q000528:radiation effects; D017421:Sequence Analysis; D014357:Trypsin / Q000378:metabolism; D014466:Ultraviolet Rays
D000345:Affinity Labels; D001386:Azides; D009711:Nucleotides; D010446:Peptide Fragments; C042231:2-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin; D006180:Proton-Translocating ATPases