Isolation and characterization of a photoaffinity-labeled peptide from the catalytic site of prenyltransferase.
Previously we presented evidence for the selective modification of the catalytic site of prenyltransferase by photoaffinity labeling with o-azidophenylethyl pyrophosphate [Brems, D. N., & Rilling, H. C. (1979) Biochemistry 18, 860]. In the present work, we report the isolation and characterization of a CNBr fragment of 30 amino acid residues from the photoaffinity-labeled enzyme. This CNBr fragment contains over 809% of the total label attached to prenyltransferase as a result of photoaffinity labeling. Several lines of evidence indicate that a number of residues in this CNBr fragment have been modified. First, Edman degradation of this labeled peptide demonstrates that at least 16 of the 30 amino acids have been modified by the photoaffinity reagent. The two most extensively modified amino acids are a specific arginine and alanine. Second, two-dimensional chromatography of Pronase digestions of the labeled CNBr fragment indicates that at least 11 different products resulted from photoaffinity labeling. Third, peptide maps of a trypsin digest of this CNBr fragment show that the attached affinity label is distributed among at least three of the resulting products of tryptic hydrolysis. Finally, comparison of amino acid analysis of this CNBr fragment with that of its counterpart isolated from native enzyme is consistent with the modification of a number of amino acids rather than a few y the photoaffinity labeling process.
D000345:Affinity Labels / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D001386:Azides / Q000378:metabolism*; D001665:Binding Sites; D002384:Catalysis; D003488:Cyanogen Bromide; D004122:Dimethylallyltranstransferase / Q000032:analysis*; D010446:Peptide Fragments / Q000302:isolation & purification*; D011402:Pronase / Q000378:metabolism; D014166:Transferases / Q000032:analysis*; D014357:Trypsin / Q000378:metabolism
D000345:Affinity Labels; D000596:Amino Acids; D001386:Azides; D010446:Peptide Fragments; C018842:2-azidophenethyl pyrophosphate; D014166:Transferases; D004122:Dimethylallyltranstransferase; D014357:Trypsin; D011402:Pronase; D003488:Cyanogen Bromide