Studies on the mechanism of action of the histone kinase dependent on adenosine 3',5'-monophosphate. Interaction of ATP with the catalytic subunit of the pig-brain enzyme: application of the quenched-flow technique

A significant release of inorganic phosphate from ATP in the presence of the cAMP-dependent pig-brain histone kinase was detected. The high degree of homogeneity of the enzyme preparations used, identity of Michaelis constants (Km for ATP = 12 microM), the close values of cAMP activation constants (48 nM and 51 nM) for the phosphotransferase and ATPase activities, respectively, are all evidence that ATP decomposition is catalysed by the histone kinase under study. The ATPase activity observed supports the ping-pong bi-bi mechanism established earlier for the phosphotransferase reaction and can be regarded as due to decomposition of the phosphoryl enzyme. The transient and steady-state phases of the ATP hydrolysis were studied. The simplest reaction pathway can be described in terms of a three-step mechanism. The close values of the rate constant for the elementary stages of the ATPase reaction obtained in the nucleophile competition study and by computer simulation of the quenched-flow kinetics give further support for the mechanism proposed. The phosphoryl enzyme decomposition was shown to be a rate-limiting step under the experimental conditions used (pH 7.8-8.0).