Phosphofructokinase from baker's yeast: studies on the initial kinetics and the fast reacting thiol groups of a...

The initial kinetics as well as the reactivity of the fast reacting thiol groups of a tetrameric form of phosphofructokinase from baker's yeast (called 12 S-enzyme), obtained by limited proteolysis in the presence of ATP were studied by the stopped-flow technique. Before attaining the steady state, the reaction shows a lag phase in the product formation, the duration of which decreases with increasing enzyme concentration. The lag phase disappears after preincubation of the enzyme with either fructose 6-phosphate, fructose 1,6-bisphosphate or fructose 2,6-bisphosphate. The occurrence of an initial transient phase suggests that the enzyme converts from a state of low activity into a highly active one after starting the reaction. The modified enzyme was found to contain two fast reacting cysteinyl residues with respect to their reactivity towards 5,5'-dithiobis(2-nitrobenzoic acid). Fructose 6-phosphate, fructose 1,6-bisphosphate and fructose 2,6-bisphosphate, respectively, decrease the reactivity of this class of thiol groups but not the total number of titrable cysteins. This result supports the hypothesis of a conformational change as a consequence of the effector binding.