Comparison of the sulfur-oxygenation of cysteine and S-carboxymethyl-l-cysteine in human hepatic cytosol and the rôle of cysteine dioxygenase

Objectives: To determine the K_m , V_max , cofactor, activator and inhibitor requirements of human cysteine dioxygenase and S-carboxymethyl-l-cysteine S-oxygenase with respect to both l-Cysteine and S-carboxymethyl-l-cysteine as substrates.

Methods: In vitro human hepatic cytosolic fraction Enzyme assays were optimised for cysteine dioxygenase activity using l-Cysteine as substrate and the effect of various cofactors, activators and inhibitors on the S-oxidations of both l-Cysteine and S-carboxymethyl-l-cysteine were investigated.

Key findings: The results of the in vitro reaction phenotyping investigation found that although both cysteine dioxygenase and S-carboxymethyl-l-cysteine S-oxygenase required Fe²⁺ for catalytic activity both enzymes showed considerable divergence in cofactor, activator and inhibitor specificities. Cysteine dioxygenase has no cofactor but uses NAD⁺ and NADH(H⁺ ) as pharmacological chaperones and is not inhibited by S-carboxymethyl-l-cysteine. S-carboxymethyl-l-cysteine S-oxygenase requires tetrahydrobiopterin as a cofactor, is not activated by NAD⁺ and NADH(H⁺ ) but is activated by l-Cysteine. Additionally, the sulfydryl alkylating agent, N-ethylmaleimide, activated carboxymethyl-l-cysteine S-oxygenase but inhibited cysteine dioxygenase.

Conclusions: Human hepatic cytosolic fraction cysteine dioxygenase activity is not responsible for the S-oxidation of the substituted cysteine, S-carboxymethyl-l-cysteine.