Fructose diphosphate aldolase of yeast: studies on the sulfhydryl groups

Abstract

Yeast fructose 1,6-diphosphate aldolase is inhibited by mercury ion and p-hydroxymercuribenzoate. The respective inhibitions are freely reversed upon the addition of thiol. Incubation of the enzyme at values as low as pH 4.5 inhibited activity, and the inactivated enzyme was reactivated by Cleland's reagent. The results suggested that this mode of inhibition proceeded initially via disulfide bond formation.Quantitative –SH group titrations of the native enzyme indicate that approximately six residues are available for reaction. The addition of 6 M urea increased the available –SH groups to between eight and nine. This latter value approaches the number of half-cystine residues determined following amino acid analysis of the native enzyme. Quantitative –SH group titrations with 5,5′-dithiobis(2-nitrobenzoic acid) also indicated four to five available residues, while in the presence of 6 M urea an additional four residues were released. Chelate inhibitors of the enzyme, EDTA and o-phenanthroline, behaved in a manner similar to 6 M urea in that additional –SH groups were released for reaction. Qualitative rate studies showed that the first group of four –SH residues reacted at least 100 times more rapidly than the second group. The significance of these two classes of –SH residues to catalysis and structure is discussed.