Identification of the sites of modification on bovine carbonic anhydrase II (BCA II) by the salt-bridge reagent cynaogen, C2N2

G. Ghenbot, T. Emge, R. A. Day

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4 Scopus citations


the hydrolase activities of bovine carbonic anhydrase B (BCA II carbonate hydrolyase, EC were modified by cyanogen (C2N2, NCCN, ethanedinitrile) with decreases in Fmax of as much as 99%. This was not accompanied by a reduction in hydrolase activity. These changes were not revrsed at lower pH values but the enzymatic activity was restored by incubation at pH 10. 14C-labeled glycine ethyl ester ([14C]GEE) specifically and covalently bound to the cyanogen-treated BCA II, as verified by HPLC and 14C monitoring. It was shown that sites of cyanogen-introduced modifications in BCA II could be effectively labeled and identifie by incubation with the nucleophile [14C]GEE. Three radiolabeled tryptic peptides from BCA II arising from a labeling process designed to study cyanogen-induced modifications leading to nucleophile labile covalent bonds have been isolated. The residues identified by ]14C]GEE labeling were Asp-34, Glut-117 and Asp-152. Three moieties attached to the ω-carboxyls by C2N2 were tentatively identified by molecular modeling; they were Arg-111, His-107 and/or His-119 and Ser-216, respectively. The use of C2N2 afforded a means to compare the salt bridges in two species and showed that two of three were not conserved.

Original languageEnglish (US)
Pages (from-to)59-65
Number of pages7
JournalBiochimica et Biophysica Acta (BBA)/Protein Structure and Molecular
Issue number1
StatePublished - Jan 15 1993
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Structural Biology
  • Biochemistry
  • Molecular Biology


  • Carbonic anhydrase H
  • Cyanogen
  • Molecular modeling
  • Salt bridge reagent


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