5'-[p-(Fluorosulfonyl)benzoyl]adenosine (FSBA) inactivates the catalytic subunit of the adenosine cyclic 3',5'-monophosphate dependent protein kinase isolated from bovine cardiac muscle by covalent modification of lysine-71, whereas 7-chloro-4-nitro-2,1,3-benzoxadiazole (NBD-Cl) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) react with cysteines-199 and -343 to inactivate the enzyme. All three of these reagents have been postulated to modify residues at or near the active site of the catalytic subunit. ATP (2 mM) in the presence of excess Mg2+ (10 mM) protects the enzyme against inactivation by these reagents. AMP did not afford any protection, but adenosine slightly decreased the rate of inactivation. The specific effects of covalent modification of lysine-71 and cysteines-199 and -343 on nucleotide binding were characterized by fluorescence-polarization titrations with lin-benzoadenine nucleotides as fluorescent ligands. lin-Benzoadenosine is a competitive inhibitor of the catalytic subunit with respect to ATP with a Ki (38 microM) similar to the Ki for adenosine (35 microM). This value agrees well with the Kd (32 microM) for adenosine determined by fluorescence-polarization titrations. lin-Benzoadenosine 5'-diphosphate (lin-benzo-ADP) has been shown to be a competitive inhibitor with respect to ATP [Hartl, F. T., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 2347], and lin-benzoadenosine 5'-triphosphate (lin-benzo-ATP) is a substrate for the phosphotransferase activity of the protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)