Chemical identification of serine 181 at the ATP-binding site of myosin as a residue esterified selectively by the fluorescent reagent 9-anthroylnitrile.
The esterification reagent 9-anthroylnitrile (ANN) reacts with a serine residue in the NH2-terminal 23-kDa peptide segment of myosin subfragment-1 heavy chain to yield a fluorescent S1 derivative labeled by the anthroyl group (Hiratsuka, T. (1989) J. Biol. Chem. 264, 18188-18194). The labeling was highly selective and accelerated by nucleotides. In the present study, to determine the exact location of the labeled serine residue, the labeled 23-kDa peptide fragment was isolated. The subsequent extensive proteolytic digestion of the peptide fragment yielded two labeled peptides, a pentapeptide and its precursor nonapeptide. Amino acid sequence and composition analyses of both labeled peptides revealed that the anthroyl group is attached to Ser-181 involved in the phosphate binding loop for ATP (Smith, C. A., and Rayment, I. (1996) Biochemistry 35, 5404-5417). We concluded that ANN can esterify Ser-181 selectively out of over 40 serine residues in the subfragment 1 heavy chain. Thus ANN is proved to be a valuable fluorescent tool to identify peptides containing the phosphate binding loop of S1 and to detect the conformational changes around this loop.
Hiratsuka
Toshiaki
T
Department of Chemistry, Asahikawa Medical College, Asahikawa 078-8510, Japan. toshiaki@asahikawa-med.ac.jp
Katoh
Tsuyoshi
T
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D000873:Anthracenes / Q000737:chemistry*; D001665:Binding Sites; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004951:Esterification; D005456:Fluorescent Dyes / Q000737:chemistry*; D006868:Hydrolysis; D009218:Myosins / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D012694:Serine / Q000378:metabolism*; D013057:Spectrum Analysis
D000873:Anthracenes; D005456:Fluorescent Dyes; D012694:Serine; C066291:9-anthroylnitrile; D000255:Adenosine Triphosphate; D009218:Myosins