Cation requirements for the acetic thiokinase from yeast

• 1.

  1. Acetic thiokinase (acetate: CoA ligase (AMP), EC 6.2.1.1) has been partially purified from yeast and the effects of Mg²⁺ and univalent cations on the overall reaction catalyzed by the enzyme and the individual steps of the reaction have been investigated. In addition of Mg²⁺ the dialyzed enzyme required K⁺, NH₄⁺ or Rb⁺ for the catalysis of the overall reaction. The concentrations of univalent cations required for optimum activity were in the range from 0.01 to 0.05 M. The addition of Cs⁺ to reaction mixtures resulted in an intermediate stimulation of activity whereas Na⁺ or Li⁺ were relatively ineffective.

• 2.

  2. It has been confirmed that the first step of the overall acetic thiokinase reaction, assayed by determining the rate of synthesis of ATP from adenyl acetate and Tris pyrophosphate, requires Mg²⁺. This reaction does not require univalent cations. In contrast, the second step of the overall reaction in which acetyl-CoA and AMP are synthesized from adenyl acetate and CoA, requires univalent cations but is inhibited by Mg²⁺ at a concentration of 0.01 M.

• 3.

  3. In most experiments it has not been possible to dialyze the yeast acetic thiokinase sufficiently to show an absolute univalent cation requirement for either the overall reaction or the step in which acetyl-CoA and AMP are synthesized from adenyl acetate and CoA. Dialyzed enzyme extracts generally contain from 6·10⁻³ to 8·10⁻³ M K⁺ which is sufficient to account for the activity in the absence of added univalent cations.

• 4.

  4. The addition of KCl to the dialyzed acetic thiokinase greatly influences the maximum velocity of the reaction in which acetyl-CoA and AMP are formed from adenyl acetate and CoA. The addition of KCl to reactions, however, fails to measurably affect the K_m values for either adenyl acetate or CoA. It is concluded that KCl influences either the rate of breakdown of an enzyme-adenyl acetate-CoA complex or the number of active enzyme sites involved in the synthesis of acetyl-CoA and AMP from adenyl acetate and CoA.