Topography of all tyrosine residues in subtilisin DY.
The extracellular alkaline proteinase subtilisin DY was nitrated with increasing amounts of tetranitromethane. At 2-fold molar excess of the reagent with respect to the tyrosine residues in the enzyme, when 1.3 residues were modified, a peak of the caseinolytic activity (13% increase) was observed. Evidence is provided that the diminishing of the pK of the phenolic hydroxyl group in Tyr(3NO2)104 causes this phenomenon. The products obtained after nitration of the enzyme with 5-fold and 200-fold molar excess of tetranitromethane were cleaved by trypsin and cyanogen bromide and the peptides obtained were studied by analysis with respect to the tyrosine and 3-nitrotyrosine residues. Their degree of substitution was established. Tyrosine-104 was the first modified residue, then follow the residues with numbers 57, 143, 206, 262 and somewhat later 21, 209, 263, all fully modified by 200-fold molar excess of the reagent. Partial modification was observed at numbers 91, 167, 214, 238 and no modification at numbers 6 and 171. It has been established that the nonmodified residues are buried inside the molecule and the partially modified residues are screened by the side chains of lysine, valine, leucine, and tryptophan as seen on a working video three-dimensional model of subtilisin Carlsberg. The approach for characterization of tyrosyl groups in proteins based on peptide sequencing and HPLC quantitation of the phenylthiohydantoin derivatives of tyrosine and 3-nitrotyrosine was further developed with respect to the quantitation of the HPLC-separated peptides using fragments of the protein studied.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002942:Circular Dichroism; D003488:Cyanogen Bromide; D008697:Methane / Q000031:analogs & derivatives*; D010449:Peptide Mapping; D011485:Protein Binding; D011487:Protein Conformation; D013381:Subtilisins / Q000378:metabolism*; D013774:Tetranitromethane / Q000378:metabolism*; D014357:Trypsin; D014443:Tyrosine
D000596:Amino Acids; D014443:Tyrosine; D013381:Subtilisins; D014357:Trypsin; D013774:Tetranitromethane; D008697:Methane; D003488:Cyanogen Bromide