Specific Irreversible Inhibition of Enzymes Concomitant to the Oxidation of Carbanionic Enzyme · Substrate Intermediates by Hexacyanoferrate(III)

Four different enzymes, class I fructose‐1,6‐bisphosphate aldolase from rabbit muscle, class II fructose‐l,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). The rate of inactivation is independent of the concentration of the enzyme, suggesting that the inactivation results from an intramolecular reaction, i.e. the inactivating agent produced by oxidation of the enzyme · substrate intermediate is not released from the active site but rather reacts in situ with the enzyme protein. Accordingly, addition of fructose 1,6‐bisphosphate to a mixture of aldolase and transaldolase (or transketolase) with hexacyanoferrate(III) initiates the exclusive inactivation of aldolase while the addition of fructose 6‐phosphate induces the exclusive inactivation of transaldolase (or transketolase respectively). The combination of natural substrate with hexacyanoferrate(III) thus constitutes a binary system for the chemical modification of enzymes that form oxidizable carbanionic intermediates. The modification by oxidative activation of enzyme · substrate intermediates is (a) highly specific for a given enzyme (the degree of specificity is determined by the binding and kinetic specificity of the enzyme for its natural substrate) and (b) results in a modification of groups at or near the enzyme's active site (reaction of the oxidatively activated substrate in situ ).