Effect of cadmium, mercury and copper on partially purified hepatic flavokinase of rat

Abstract

The effect of cadmium (Cd²⁺), mercury (Hg²⁺) and copper (Cu²⁺) was studied with partially purified flavokinase (ATP:riboflavin 5-phosphotransferase EC 2.7.1.26) from rat liver. All the divalent heavy metal cations inhibited flavokinase activity in a concentration-dependent manner. The inhibitory effect of cadmium on the enzyme was completely reversed by increasing concentration, of Zinc (Zn²⁺) indicating a competition between Zn²⁺ and Cd²⁺ for binding with the enzyme. A competition between riboflavin and Cd2+ is also evident from the present investigation. These observations hint at the possibility that Zn²⁺ and Cd²⁺ probably compete for the same site on the enzyme where riboflavin binds. However, inhibition of flavokinase by Hg²⁺ could not be reversed by Zn²⁺. Our studies further reveal that hepatic flavokinase appears to contain an essential, accessible and functional thiol group(s) which is evident from a concentration dependent inhibition of activity by sulfhydryl reagent s like parachloromercuribenzoate (PCMB), 5,5-dithiobis (2-nitrobenzoic acid)(DTNB), and N-ethylmaleimide (NEM). Inhibition of flavokinase by sulfhydryl reagents were protected, except in case of NEM inhibition, when the enzyme was incubated with thiol protectors like glutathione (GSH) and dithiothreitol (DTT). Furthermore, the enzyme could also be protected from the inhibitory effect of Cd²⁺ and Hg²⁺ by GSH and DTT suggesting that Cd²⁺ probably interacts with a reactive thiol group at or near the active site of enzyme in bringing about its inhibitory effect.