Amino acid sequence of the active site of Acanthamoeba myosin II.
We have used the substrate [5,6-3H]UTP for direct photoaffinity labeling of the active site of the heavy chain of myosin II from Acanthamoeba castellanii. The only labeled peptide in a total tryptic digest had the sequence of Thr-Glu-Asn-Thr-Me2Lys-Lys (where Me2Lys represents dimethyllysine) with the substrate covalently bound to the Glu residue. This sequence differs at only one position from the sequence of residues 184-189 of nematode myosin heavy chain (Me2Lys----Lys), a post-translational modification, and at two additional positions from residues 185-190 of rabbit skeletal muscle myosin (Glu----Val and Lys----Arg). The partial sequence of a larger labeled peptide derived from total chymotryptic digestion was compatible with and extended this sequence. A 20-residue sequence that contains the active site, tryptic hexapeptide is otherwise identical in Acanthamoeba and rabbit skeletal muscle myosins and has only one more difference in nematode myosin. Because UTP is a substrate for myosin II and a "zero-length" probe, we believe that it identifies amino acid residues that are very close to the substrate during the catalytic cycle.
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000656:Amoeba / Q000032:analysis*; D000818:Animals; D001665:Binding Sites; D002918:Chymotrypsin; D009218:Myosins / Q000032:analysis*; D009348:Nematoda / Q000032:analysis; D010455:Peptides / Q000032:analysis; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity; D014357:Trypsin; D014544:Uridine Triphosphate / Q000378:metabolism
D000345:Affinity Labels; D010455:Peptides; D002918:Chymotrypsin; D014357:Trypsin; D009218:Myosins; D014544:Uridine Triphosphate