Probe of subunit structure in fungal p-diphenol oxidase by treatment with guanidine hydrochloride and at high pH

Abstract

The possible subunit structure of the copper-containing p-diphenol oxidase ( p-diphenol:O 2 oxidoreductase, EC 1.10.3.2, formerly known as laccase from Polyporus versicolor was investigated by treating the enzyme in guanidine·HC1 plus thiol at pH 5.5 and 8.6, and in pH 12.5 borate before and after succinylation. As the guanidine·HCl concentration is raised, extensive unfolding is produced between 2.5 and 3.5–4 M. (Some structural rearrangements affecting aromatic residues and disulfide groups, as well as Cu ions, occur at guanidine·HCl concentration below the range controlling the large unfolding; and at 2 M or higher, added thiol must be supplied to prevent aggregation due to sulfhydryl disulfide interchange.) p-Diphenol oxidase in 6 M guanidine·HCl plus thiol, or the completely S-carboxymethylated protein without thiol, is fragmented to a relatively homogeneous mixture displaying weight-average molecular weight between the native value and half that value; this is interpreted as meaning partial dissociation subject to chemical equilibrium. Treatment at pH 12.5, however, fragments p-diphenol oxidase to the level of half-molecules and succinylated p-diphenol oxidase to the level of quarter-molecules. (Aggregation, due to sulfhydryl-disulfide interchange, is prevented if the proteins have been completely S-carboxymethylated.) Cleavage of a very few peptide bonds is not ruled out, although random cleavage of a significant number of peptide or other covalent bonds is excluded. These fragmentations, also, may be considered to operate by dissociation subject to chemical equilibrium; alternate interpretation as cleavage of peptide or other covalent bonds, ( viz., other than disulfide bonds) requires that such cleavage be quite specific. The results are explained by a p-diphenol oxidase structure consisting of four parts of similar size—it is of considerable interest that this is compatible with the known binding in p-diphenol oxidase of 4 Cu ions per native enzyme molecule, 2 Cu(I), and 2 Cu(II) with different properties. The results favor existence of separate polypeptide chains, linked by disulfide groups and noncovalent interactions.