title: 6-Mercapto-FAD and 6-thiocyanato-FAD as active site probes of phenol hydroxylase.
abstract: Recently, the synthesis and properties of several 6-substituted flavins as active site probes for flavoproteins have been reported (Ghisla, S., Massey, V., and Yagi, K. (1986) Biochemistry 25, 3282-3289). Here, we report results of experiments in which 6-thiocyanato-FAD and 6-mercapto-FAD have been substituted for the native flavin of phenol hydroxylase. The 6-SCN-FAD enzyme was converted spontaneously to the 6-mercaptoflavin form probably due to dissociation of flavin, followed by attack of external protein thiols. The pK alpha values of uncomplexed and phenol-bound 6-mercapto-FAD enzyme were determined. Both the spontaneously formed 6-mercapto-FAD enzyme and the enzyme reconstituted with preformed 6-mercapto-FAD were treated with a variety of thiol-specific reagents, and reaction rates were followed by spectroscopic means. Comparison with the corresponding rates found with free flavin suggested a high degree of accessibility to the flavin 6-position. Accessibility was somewhat decreased in the presence of phenol. Upon treatment with low concentrations of methyl methanethiosulfonate or N-ethylmaleimide (NEM), extremely rapid spectral changes were apparent. The former reaction, however, was reversed spontaneously within 2 h. Reaction with NEM was biphasic, with spectral changes consistent with the mechanism previously proposed (Steenkamp, D. J., McIntire, W., and Kenney, W. C. (1978) J. Biol. Chem. 253, 2818-2824), followed by a small absorbance decrease due to protein conformational changes. The NEM reaction is unusual, being easily reversed by addition of excess dithiothreitol.
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
authors.forename: M G
authors.initials: MG
authors.lastname: Taylor
authors.forename: V
authors.initials: V
authors.lastname: Massey
mesh_terms: D001665:Binding Sites; D002723:Chlorobenzoates / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D005182:Flavin-Adenine Dinucleotide / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008741:Methyl Methanesulfonate / Q000031:analogs & derivatives / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D015335:Molecular Probes; D015394:Molecular Structure; D010636:Phenols / Q000378:metabolism; D013057:Spectrum Analysis
chemical_list: D002723:Chlorobenzoates; D015335:Molecular Probes; D010636:Phenols; C068703:6-thiocyanato-FAD; C068704:6-mercapto-FAD; D005182:Flavin-Adenine Dinucleotide; C014674:methyl methanethiosulfonate; D008741:Methyl Methanesulfonate; D006861:Hydrogen Peroxide; D006899:Mixed Function Oxygenases; C022336:phenol 2-monooxygenase; C000433:3-chloroperbenzoic acid; D005033:Ethylmaleimide; D004229:Dithiothreitol