nitrophenols and glycine-ethyl-ester

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.

Topics: Amino Acids; Animals; Binding Sites; Carbonic Anhydrases; Cattle; Chromatography, High Pressure Liquid; Glycine; Hydrogen-Ion Concentration; Kinetics; Models, Molecular; Nitriles; Nitrophenols; Trypsin