Sulphydryl oxidase: oxidation of sulphydryl groups and the formation of three-dimensional structure in proteins

Abstract

Sulphydryl oxidase, an enzyme isolated from milk, catalyses the de novo synthesis of disulphide bonds. Thiol groups in amino acids or their derivatives, peptides, and proteins are oxidized; molecular oxygen serves as the electron acceptor and undergoes a two-electron reduction to hydrogen peroxide. Michaelis constants vary considerably amongst various substrates; glutathione is a particularly good substrate. Inhibition studies and oxidation of 1,3-diphenylisobenzofuran suggest a mechanism involving an electron transfer to singlet O2 forming an enzyme-bound hydroperoxy group. Evidence for a direct interaction of the enzyme with horseradish peroxidase was also obtained. Although protein-folding appears to be thermodynamically favoured, rates of spontaneous acquisition of functional three-dimensional structures in disulphide-containing proteins have appeared disturbingly slow. In the presence of sulphydryl oxidase, functional structure is rapidly acquired by both reductively unfolded ribonuclease A and reductively denatured immobilized chymotrypsinogen A as judged by restoration of native fluorescence characteristics and biological activity. Preliminary data suggest that unlike thiol:protein-disulphide oxidoreductase, protein-disulphide isomerase, or GSSG/GSH redox systems, sulphydryl oxidase does not permit a 'reshuffling' of disulphide bonds.