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A Deoxyribonuclease Which Requires Nucleoside Triphosphate from Micrococcus lysodeikticus

III. EXCHANGE REACTION CATALYZED BY THE ENZYME PREPARATION

From the ¹ From the Department of Biochemistry, Kyushu University, Faculty of Medicine, Fukuoka, Japan

The ADP-ATP exchange reaction catalyzed by the highly purified Micrococcus lysodeikticus DNase has been investigated in detail. The exchange activity is eluted together with the DNase activity from triethylaminoethylcellulose and the ratio between both activities is relatively constant in the peak fractions. The exchange reaction between [8-¹⁴C]ADP and ATP has an absolute requirement for ATP and divalent cations. The enzyme is not dependent on the presence of a DNA substrate. The enzyme does not catalyze a measurable exchange between ATP and orthophosphate. The optimum pH of the reaction is 8.5 in glycine-NaOH buffer. Mn²⁺ is the most effective metal ion but can be replaced, to some extent, by Mg²⁺, Co²⁺, or Ni²⁺. Of the nucleoside triphosphates tested, ATP is most effective in the presence of Mn²⁺, while in the presence of Mg²⁺ instead of Mn²⁺, ATP and dATP are most effective at almost the same level. In contrast to the ADP saturation curve which shows typical Michaelis-Menten kinetics, the ATP saturation curve is sigmoidal, suggesting multiple binding of ATP on the enzyme. These results suggest that the enzyme and ATP react to form the enzyme-phosphate intermediate as the first step of the over-all reaction of DNA hydrolysis.

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