Specific irreversible inhibition of enzymes concomitant to the oxidation of carbanionic enzyme-substrate intermediates by hexacyanoferrate (III)

Abstract

Four different enzymes, class I fructose-1,6-biphosphate aldolase from rabbit muscle, class II fructose-1,6-bisphosphate aldolase from yeast, transaldolase, and transketolase, are inactivated progressively in the presence of their specific substrates and hexacyanoferrate(III). The inactivation is strictly linked to the oxidation of the carbanionic enzyme-substrate intermediates of these enzymes reported previously [Healy, M. J. and Christen, P. (1973) Biochemistry, 12, 35]. However, the loss of activity is not due to the products of this oxidation, i.e. to hexacyanoferrate(II), or to the oxidation product of the substrate such as hydroxypyruvaldehyde phosphate in the case of aldolase [Healy, M. J. and Christen, P. (1972) J. Am. Chem. Soc. 94, 7911]. The inactivation is not reversed on removal of low-molecular-weight compounds by gel filtration or extensive dialysis indicating a covalent modification of the enzyme. The rate of inactivation obeys saturation kinetics with respect to substrate concentration. Hence, the modifying agent is a transiently reactive intermediate formed during the oxidation of the carbanionic enzyme-substrate intermediate by hexacyanoferrate(III).