Affinity labeling of adenylate kinase with adenosine diphosphopyridoxal. Presence of Lys21 in the ATP-binding site.
Adenosine diphosphopyridoxal, the affinity labeling reagent specific for a lysyl residue in the nucleotide-binding site of several enzymes (Tagaya, M., and Fukui, T. (1986) Biochemistry 25, 2958-2964; Tamura, J. K., Rakov, R. D., and Cross R. L. (1986) J. Biol. Chem. 261, 4126-4133) was applied to adenylate kinase from rabbit muscle. Incubation of the enzyme with a low concentration of the reagent at 25 degrees C for 20 min followed by reduction by sodium borohydride resulted in rapid inactivation of the enzyme. Extrapolation to 100% loss of enzyme activity gave a value of 1.0 mol of the reagent per mol of enzyme. ADP, ATP, and MgATP almost completely protected the enzyme from inactivation, whereas AMP offered little retardation of the inactivation. Dilution of the inactivated enzyme which had not been treated with the reducing reagent led to restoration of enzyme activity. This reactivation was accelerated by ATP but not by AMP. Structural study of the labeled peptide showed that Lys21 is exclusively labeled by adenosine diphosphopyridoxal. These results suggest that the epsilon-amino group of Lys21 is located in the ATP-binding site of the enzyme, more specifically at or close to the subsite for the gamma-phosphate of the nucleotide.
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000263:Adenylate Kinase / Q000378:metabolism*; D000345:Affinity Labels; D000818:Animals; D001665:Binding Sites; D007700:Kinetics; D008239:Lysine; D009132:Muscles / Q000201:enzymology; D010770:Phosphotransferases / Q000378:metabolism*; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives*; D011817:Rabbits
D000345:Affinity Labels; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010770:Phosphotransferases; D000263:Adenylate Kinase; C048236:pyridoxal 5'-diphospho-5'-adenosine; D008239:Lysine