Identification of the S-adenosyl-L-methionine binding site of protein-carboxyl O-methyltransferase using 8-azido-S-adenosyl-L-methionine.
Protein-carboxyl O-methyltransferase (protein methylase II) transfers the methyl group from S-adenosyl-L-methionine (AdoMet) to the carboxyl side chains of the amino acids in the proteins. We have used the radiolabeled analogue of AdoMet, 8-azido-S-adenosyl-L-[methyl-3H]methionine (8-N3-Ado[methyl-3H]Met), to investigate the AdoMet binding site of protein methylase II. The incorporation of the photoaffinity label in the enzyme upon UV irradiation is highly specific. In the absence of UV irradiation or if the photoprobe is irradiated prior to its addition to the reaction mixture, no photoinsertion of the label occurs. Moreover, the presence of a competitive inhibitor of protein methylase II, S-adenosyl-L-homocysteine (AdoHcy), or the unlabeled AdoMet itself in the reaction mixture diminished labeling of the enzyme. Sequential digestion of the labeled enzyme with trypsin, chymotrypsin, and endoproteinase Glu-C yielded a modified and radiolabeled decapeptide. When compared with the reported primary amino acid sequence of protein methylase II from rat brain, the amino acid composition of the decapeptide matched residues 113-121. This segment forms the midpoint region of the enzyme (234 amino acid residues). An important characteristic of the sequence is the presence of two adjacent aspartic acid residues (Asp117-Asp118) which most likely provide the negative charge environment for the sulfonium moiety of the AdoMet molecule.
Syed
S K
SK
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Kim
S
S
Paik
W K
WK
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001386:Azides; D001665:Binding Sites; D002417:Cattle; D008969:Molecular Sequence Data; D010777:Photochemistry; D011497:Protein O-Methyltransferase / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D012436:S-Adenosylmethionine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D000345:Affinity Labels; D000596:Amino Acids; D001386:Azides; D012436:S-Adenosylmethionine; C037207:8-azido-S-adenosylmethionine; D011497:Protein O-Methyltransferase