Kochetkov S N, Bulargina T V, Sashchenko L P, Severin E S
Eur J Biochem. 1977 Nov 15;81(1):111-8. doi: 10.1111/j.1432-1033.1977.tb11932.x.
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.
研究了依赖于腺苷3':5'-单磷酸（环磷酸腺苷）的组蛋白激酶催化的磷酸残基转移反应。磷酸转移酶反应显示遵循乒乓双底物类型机制。用[γ-32P]ATP孵育组蛋白激酶的催化亚基后，观察到每摩尔蛋白质掺入一摩尔[32P]磷酸基团。分离出含胰蛋白酶[32P]磷酸组氨酸的肽段，并测定其N端和氨基酸组成。ATP的2',3'-二醛衍生物（oATP）用作环磷酸腺苷依赖性组蛋白激酶催化亚基的亲和标记物。该抑制剂与活性位点赖氨酸残基的ε-氨基形成醛亚胺键，经硼氢化钠还原后不可逆地与酶结合，同时酶发生不可逆失活。失活后，每摩尔酶掺入约一摩尔14C标记的抑制剂。ATP有效地保护组蛋白激酶的催化亚基不被oATP失活。对酶-抑制剂复合物进行胰蛋白酶消化，得到了组蛋白激酶活性位点的14C标记肽段。基于这些结果，推测了组蛋白激酶催化亚基活性位点中组氨酸和赖氨酸残基的作用。
