Studies on the mechanism of action of histone kinase dependent on adenosine 3':5'-monophosphate. Evidence for involvement of histidine and lysine residues in the phosphotransferase reaction.
The reaction of the phosphate residue transfer catalysed by histone kinase dependent on adenosine 3':5'-monophosphate (cyclic AMP) was studied. The phosphotransferase reaction was shown to obey the mechanism of ping-pong bi-bi type. After incubation of the catalytic subunit of histone kinase with [gamma-32P]ATP the incorporation of one mole of [32P]phosphage per mole of protein was observed. The tryptic [32P]phosphohistidine-containing peptide was isolated and its N-terminus and amino acid composition were determined. The 2',3'-dialdehyde derivative of ATP (oATP) was used as the affinity label for the catalytic subunit of cyclic-AMP-dependent histone kinase. The inhibitor formed an alidmine bond with epsilon-amino group of the lysine residue of the active site and was irreversibly bound to the enzyme after reduction by sodium borohydride with concurrent irreversible inactivation of the enzyme. After inactivation, about one mole of 14C-labelled inhibitor was incorporated per mole of the enzyme. ATP effectively protected the catalytic subunit of histone kinase against inactivation by oATP. Tryptic digestion of the enzyme-inhibitor complex led to the isolation of the 14C-labelled peptide of the active site of histone kinase. Basing on these results, the role of histidine and lysine residues in the active site of the catalytic subunit of histone kinase was suggested.
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D000242:Cyclic AMP / Q000494:pharmacology*; D004789:Enzyme Activation; D006639:Histidine; D007700:Kinetics; D008239:Lysine; D010446:Peptide Fragments / Q000032:analysis; D006656:Protamine Kinase / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D014357:Trypsin
D000596:Amino Acids; D010446:Peptide Fragments; D006639:Histidine; D000242:Cyclic AMP; D011494:Protein Kinases; D006656:Protamine Kinase; D014357:Trypsin; D008239:Lysine