Serine-324 of myosin's heavy chain is photoaffinity-labeled by 3'(2')-O-(4-benzoylbenzoyl)adenosine triphosphate.
A portion of the active site of rabbit skeletal myosin near the ribose ring of ATP can be labeled by the photoaffinity analogue 3'(2')-O-(4-benzoylbenzoyl)adenosine triphosphate (Bz2ATP). The specificity of the photolabeling was assured by first trapping [14C]Bz2ATP at the active site by use of thiol cross-linking agents [Mahmood, R., Cremo, C., Nakamaye, K., & Yount, R. (1987) J. Biol. Chem. 262, 14479-14486]. Five radioactive peptides were isolated by high-performance liquid chromatography after extensive trypsin and subtilisin digestion of photolabeled myosin subfragment 1. Four of these peptides were sequenced by Edman techniques, and all originated from a region with the sequence Gly-Glu-Ile-Thr-Val-Pro-Ser-Ile-Asp-Asp-Gln, which corresponds to rabbit myosin heavy chain residues 318-328. The fifth labeled peptide had an amino acid composition appropriate for residues 312-328. Amino acid composition, radiochemical analysis, and sequence data indicate that Ser-324 is the major amino acid residue photolabeled by Bz2ATP. Spectrophotometric evidence indicates that the benzophenone carbonyl group has inserted into a C-H bond from either the alpha- or beta-carbon of serine. These results place Ser-324 at a distance of 6-7 A from the 3'(2') ribose oxygens of ATP bound at the active site of myosin.
Mahmood
R
R
Institute of Biological Chemistry, Washington State University, Pullman 99164-4660.
Elzinga
M
M
Yount
R G
RG
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000345:Affinity Labels / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D012694:Serine; D013056:Spectrophotometry, Ultraviolet
D000345:Affinity Labels; D000596:Amino Acids; D015879:Myosin Subfragments; D010446:Peptide Fragments; D012694:Serine; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D009218:Myosins