Studies on the fast reacting thiol groups in phosphofructokinase from baker's yeast

The fast reacting thiol groups of yeast phosphofructokinase were studied by means of stopped-flow measurements. The enzyme was found to contain four very fast reacting cysteinyl residues determined by their reactivity towards 5,5'-dithiobis(2-nitrobenzoic acid). A second class of eight thiol groups reveals an apparent first order rate constant which is three orders of magnitude lower than the rate constant of the first one. Due to the extreme high reactivity of the first class of cysteinyl residues partial oxidation was already observed under aerobic conditions. Fructose 6-phosphate, fructose 1.6-bisphosphate, and fructose 2.6-bisphosphate, respectively, decrease the reactivity of the first class of thiol groups but not the total number of the accessible cysteins. This result is discussed with regard to conformational changes of the enzyme after binding of the sugar phosphates.