Reaction of liver pyruvate kinase with sulfhydryl reagents: effect on its activity

Homogeneous liver pyruvate kinase was reacted with different sulfhydryl reagents, which included o-iodosobenzoate, 5',5'-dithiobis(2-nitrobenzoic acid) and N-ethylmaleimide. Activity determinations of the treated enzyme made with and without Fru(1,6)P2 indicate that the protein contains two sulfhydryl groups per subunit important to its properties, one more accessible than the other. Fru(1,6)P2 added to mixtures prevented loss of activity obtained with o-iodosobenzoate and 5',5'-dithiobis(2-nitrobenzoic acid). It appears that Fru(1,6)P2 does not interfere with the reaction of the reagent with the sulfhydryl group, but prevents an ensuing conformational change, which leads to changes in the enzyme's properties.