Localisation of an ATP-binding site on adenylyl cyclase type I after chemical and enzymatic fragmentation

Abstract

Photolabeling of partially purified bovine brain adenylyl cyclase (AC I) with [γ ³²P8-N-ATP led to incorporation of ³²P into the 115 kDa catalyst. Further treatment with N-chlorosuccinimide, which cleaves proteins at tryptophan residues, yielded a 14 kDA ³²P-labeled fragment. The latter was immunoprecipitated by antibody BBC1, recognizing the extreme C-terminus of AC I, but not by antibody BBC2, recognizing a more remote epitope. Further fragmentation of photolabeled AC I by the proteases Glu-C and Asp-N yielded ³²P-labeled peptides corresponding to 2.9 kDa and 5.6 kDa fragments, which were not recognized by any of these antibodies. This narrows the ATP binding site down to a 25 amino acid sequence containing a general motif G(X_0–7)KG(X_0–4)L/M(X_5–7)S/T present in all eukaryotic adenylyl cyclases so far cloned, but also in a variety of bacterial adenylyl cyclases.