Cyanocysteine-mediated molecular dissection of dihydrofolate reductase: occurrence of intra- and inter-molecular reactions forming a peptide bond.
During a cyanocysteine-mediated dissection study of dihydrofolate reductase, a peptide fragment with a molecular mass of 18 Da less than expected was found as a major reaction product when the dissection reaction was applied to a Lys-cyanocysteine linkage. Detailed characterization of the dissection products by protease digestion, peptide sequencing, liquid chromatography/electrospray ionization mass spectrometry, and capillary electrophoresis suggested that the by-product was generated via a lactam ring formation through the intramolecular nucleophilic attack of the epsilon-amino group on the carbonyl carbon of the Lys-cyanocysteine linkage. We have also demonstrated the occurrence of intermolecular attack of an alpha-amino group of glycine on the carbonyl carbon of the X-cyanocysteine linkage to form a new X-Gly linkage, which should be a useful reaction for specific modification of proteins at the C-terminal.
Takenawa
T
T
National Institute of Bioscience and Human Technology, Tsukuba, Ibaraki, 305-8566, Japan.
Oda
Y
Y
Ishihama
Y
Y
Iwakura
M
M
D001665:Binding Sites; D003545:Cysteine; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics
D013762:Tetrahydrofolate Dehydrogenase; D003545:Cysteine