Reductive alkylation of lysine residues in subtilisin DY.
Only lysine epsilon-amino groups (and the N-terminal alpha-amino group) in native subtilisin DY were reductively alkylated by glyceraldehyde in the presence of sodium cyanoborohydride. The modified protein molecule was cleaved by TosPheCH2Cl-trypsin or cyanogen bromide and the two sets of peptides obtained were fractionated and purified by gel filtration and HPLC. For determination of the degree of modification of each lysine residue, selected peptides were subjected to sequence analysis combined with quantitative estimation of the containing PTH-Lys and PTH-epsilon-DHP-Lys. The data obtained showed that the lysine residues in positions 12, 15, 27, 43, 136, 141, 265 were entirely modified, those in positions 170, 184, 237 were partially modified, and Lys22 and Lys94 were unaccessible for the reagent. The caseinolytic activity decreased by 23% when the maximum number of lysine residues (8.6 of the total 12 residues) in subtilisin DY were modified. The CD-spectra of native and modified enzyme showed only slight differences. Both these experiments suggest that the lysine residues do not take part directly in the catalytic reaction but are responsible for maintaining the native three-dimensional enzyme structure. The data obtained for the accessibility of the different lysine residues in subtilisin DY correlated very well with the positions of these residues in a video model of the structure of subtilisin Carlsberg, thus suggesting that the spatial structures of these two enzymes are very similar.
D000478:Alkylation; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002364:Caseins / Q000032:analysis; D002942:Circular Dichroism; D006868:Hydrolysis; D008239:Lysine / Q000032:analysis*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000032:analysis / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis; D013381:Subtilisins / Q000032:analysis*; D014357:Trypsin
D000596:Amino Acids; D002364:Caseins; D010455:Peptides; D011506:Proteins; D013381:Subtilisins; D014357:Trypsin; D008239:Lysine