Photochemical probes of the active site of myosin. Irradiation of trapped 3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate labels the 50-kilodalton heavy chain tryptic peptide.
3'-O-(4-Benzoyl)benzoyl-ATP (Bz2ATP), an analog of ATP containing a photoreactive benzophenone moiety, was used as a probe of the ATP binding site of myosin subfragment 1 (SF1). The inactivation of SF1 NH+4-EDTA ATPase by the bifunctional thiol crosslinking system cobalt(II)/cobalt(III) phenanthroline complexes was enhanced by Bz2ATP to the same degree as by ATP. This treatment resulted in the stable trapping of Bz2ATP at the active site in nearly stoichiometric amounts in a manner exactly analogous to ATP (Wells, J.A., and Yount, R.G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 4966-4970). Irradiation of SF1 containing trapped [3H]Bz2ATP gave approximately 50% covalent incorporation of the trapped nucleotide into the enzyme. Analysis of photolabeled SF1 by gel electrophoresis showed that all of the [3H]Bz2ATP was attached to the 95-kDa heavy chain fragment. No label was found in the light chains. Similar analysis of the same protein after limited trypsin treatment demonstrated that approximately 75% of the [3H]Bz2ATP was bound to the central 50-kDa peptide and its 75-kDa precursor from the heavy chain. The N-terminal 25-kDa tryptic peptide, shown to be photolabeled by other ATP analogs (Szilagyi, L., Balint, M., Sreter, F.A., and Gergely, J. (1979) Biochem. Biophys. Res. Commun. 87, 936-945; Okamoto, Y., and Yount, R.G. (1983) Biophys. J. 41, 298a), was not labeled (less than 1%) by Bz2ATP. These results demonstrate that portions of the 50 kDa-peptide of the heavy chain are within 6-7 A of the ATP binding site on SF1 and possibly contribute to nucleotide binding.
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism*; D010782:Photolysis; D011817:Rabbits; D014316:Tritium; D014357:Trypsin
D000345:Affinity Labels; D046911:Macromolecular Substances; D015879:Myosin Subfragments; D010446:Peptide Fragments; D014316:Tritium; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin; D000251:Adenosine Triphosphatases; D009218:Myosins