Inhibitory sites in enzymes: zinc removal and reactivation by thionein

Abstract

Thionein (T) has not been isolated previously from biological material. However, it is generated transiently in situ by removal of zinc from metallothionein under oxidoreductive conditions, particularly in the presence of selenium compounds. T very rapidly activates a group of enzymes in which zinc is bound at an inhibitory site. The reaction is selective, as is apparent from the fact that T does not remove zinc from the catalytic sites of zinc metalloenzymes. T instantaneously reverses the zinc inhibition with a stoichiometry commensurate with its known capacity to bind seven zinc atoms in the form of clusters in metallothionein. The zinc inhibition is much more pronounced than was previously reported, with dissociation constants in the low nanomolar range. Thus, T is an effective, endogenous chelating agent, suggesting the existence of a hitherto unknown and unrecognized biological regulatory system. T removes the metal from an inhibitory zinc-specific enzymatic site with a resultant marked increase of activity. The potential significance of this system is supported by the demonstration of its operations in enzymes involved in glycolysis and signal transduction.

Figure 1

Inhibition of caspase-3 by zinc. Caspase-3 (1.7 nM) was incubated with various amounts of zinc. Activity was assayed after 5 min.

Figure 2

Reactivation of glyceraldehyde 3-phosphate dehydrogenase with T in the presence of zinc. Glyceraldehyde 3-phosphate dehydrogenase (10 nM) was incubated in buffer containing 30 mM Na₂HAsO₄ with various amounts of T introduced 5 min after ZnSO₄ was added to a final concentration of 1 μM. Activities were assayed after 30 min.

Figure 3

Reactivation of zinc-inhibited glyceraldehyde 3-phosphate dehydrogenase with T. Glyceraldehyde 3-phosphate dehydrogenase (10 nM) was incubated with ZnSO₄ (1 μM) for 10 min in buffer containing 30 mM Na₂HAsO₄. Enzyme activity was measured for 0.5 min; T (250 nM) was then injected into the cuvet, and the measurement continued.