Identification of the sites of modification on bovine carbonic anhydrase II (BCA II) by the salt-bridge reagent cyanogen, C2N2.
The hydrolase activities of bovine carbonic anhydrase B (BCA II carbonate hydrolyase, EC 4.2.1.1) were modified by cyanogen (C2N2, N identical to C-C identical to N, ethanedinitrile) with decreases in Vmax of as much as 99%. This was not accompanied by a reduction in hydrolyase activity. These changes were not reversed 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 identified 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, Glu-117 and Asp-152. Three moieties attached to the omega-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.
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Department of Chemistry, University of Cincinnati, OH 45221.
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D002256:Carbonic Anhydrases / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D005998:Glycine / Q000031:analogs & derivatives / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D009570:Nitriles; D009596:Nitrophenols; D014357:Trypsin
D000596:Amino Acids; D009570:Nitriles; D009596:Nitrophenols; C022010:glycine ethyl ester; C011206:cyanogen; C008642:4-nitrophenyl acetate; D014357:Trypsin; D002256:Carbonic Anhydrases; D005998:Glycine