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

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Abstract
The reaction of the phosphate residue transfer catalyzed by pig brain histone kinase [EC 2.7.1.37] dependent on cyclic[c]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 1 mol of [32P]phosphate/mol 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 cAMP-dependent histone kinase. The inhibitor formed an aldimine 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 1 mol of 14C-labeled inhibitor was incorporated per mol 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 isolation of the 14C-labeled peptide of the active site of histone kinase. The role of histidine and lysine residues in the active site of the catalytic subunit of histone kinase was suggested.