Phenol hydroxylase from yeast. Sulfhydryl groups in phenol hydroxylase from Trichosporon cutaneum.
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 Mr 1.48 X 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'-dithio-bis(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 are 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.
D001051:Apoenzymes / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D008623:Mercaptoethanol / Q000494:pharmacology; D003904:Mitosporic Fungi / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008970:Molecular Weight; D010636:Phenols; D013438:Sulfhydryl Compounds / Q000032:analysis / Q000378:metabolism*
D001051:Apoenzymes; D006902:Hydroxymercuribenzoates; D010636:Phenols; D013438:Sulfhydryl Compounds; D005182:Flavin-Adenine Dinucleotide; D008623:Mercaptoethanol; D004228:Dithionitrobenzoic Acid; D004492:Edetic Acid; D006899:Mixed Function Oxygenases; D004229:Dithiothreitol