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

the hydrolase activities of bovine carbonic anhydrase B (BCA II carbonate hydrolyase, EC 4.2.1.1) were modified by cyanogen (C₂N₂, NCCN, ethanedinitrile) with decreases in F_max 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.