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J. Biochem, 1998, Vol. 123, No. 6 1137-1144
© 1998 Japanese Biochemical Society

research-article

Cyanocysteine-Mediated Molecular Dissection of Dihydrofolate Reductase: Occurrence of Intra- and Inter-Molecular Reactions Forming a Peptide Bond

Tatsuyuki Takenawa*, Yoshiya Oda{dagger}, Yasushi Ishihama{dagger} and Masahiro Iwakura*,1

*National Institute of Bioscience and Human Technology 1-1 Higashi, Tsukuba, Ibaraki 305-8566
{dagger}Tsukuba Research Laboratories, Eisai Co., Ltd. 5-1-3 Tokodai, Tsukuba, Ibaraki 300-26

1To whom correspondence should be addressed. E-mail: iwakura{at}nibh.go.jp

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 {varepsilon}-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.


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