Phenol Hydroxylase from Yeast

Abstract

Thiol groups in phenol hydroxylase were measured using two different –SH reagents and amino acid analysis. Stepwise blocking of the –SH groups was correlated with enzyme activity and FAD content. The results indicate that the enzyme contains 16 –SH groups per molecule of M r 1.48 × 10 5 . At least four –SH groups are not accessible without the use of a denaturing agent. There is seemingly no disulphide bridge. On the whole, the reactivity towards p ‐hydroxymercuribenzoate is much greater than towards 5,5′‐dithio‐bis(2‐nitrobenzoic acid). The two reagents seem to have a different specificity with respect to which –SH groups they attack. Either reagent dislocates FAD from the holoenzyme, leaving a characteristic mercaptide derivative of the apoenzyme. Such derivatives were used to prepare the apoenzyme. The –SH groups in the apoenzyme are much more reactive towards 5,5′‐dithiobis(2‐nitrobenzoic acid) than the –SH groups in the holoenzyme. The stoichiometry of the reaction with 5,5′‐dithio‐bis(2‐nitrobenzoic acid) indicates that at least 8 –SH groups are located in spatially close pairs. The most reactive pair of all does not appear to be of importance for enzyme activity. The two subsequent –SH pairs are essential for enzyme activity and are involved in FAD attachment.The reactivity of the –SH groups decreases dramatically in the presence of substrate, even at substrate concentrations equivalent to the level of the catalytic sites.The isolated apoenzyme has a tendency to aggregate. A large proportion of –SH groups in such aggregate(s) is buried, especially when EDTA is not used throughout the preparation of the apoenzyme. The aggregates are enzymically inactive.