A Study of the Sulfhydryl Groups of the Catalytic Subunit of Escherichia coli Aspartate Transcarbamylase: THE USE OF ENZYME-5-THIO-2-NITROBENZOATE MIXED DISULFIDES AS INTERMEDIATES IN MODIFYING ENZYME SULFHYDRYL GROUPS

Abstract

Abstract Each of the three polypeptide chains of the catalytic subunit (CSU) of aspartate transcarbamylase from Escherichia coli has a single —SH group and no disulfides. Although the —SH group is unreactive toward several S-alkylating agents and symmetrical disulfides, it reacts slowly with 5,5'-dithio-bis(2-nitrobenzoate) (DTNB) and p-hydroxymercuribenzoate. CSU is inactivated by these reagents and also, transiently, by inorganic polysulfides. Kinetic studies of the reaction with DTNB show (a) that DTNB does not bind to CSU as a prerequisite for reaction, (b) that reaction depends on the ionization of a group on the enzyme with apparent pKa = 7.9, probably the —SH group, and (c) that inhibitors of enzymatic activity also inhibit reaction of the —SH group with both DTNB and p-hydroxymercuribenzoate. Dissociation constants for phosphate and succinate determined from inhibition of the DTNB reaction agree with those previously determined by steady state kinetics and ultraviolet difference spectroscopy. Undissociated native aspartate transcarbamylase does not react with DTNB under conditions which lead to complete reaction of CSU. Treatment of the CSU-thionitrobenzoate mixed disulfide with nucleophiles such as cyanide, sulfite, and 2-mercapto-ethanol leads to new S- derivatives of CSU. The S-cyano derivative is fully active, but the S-sulfo and S-2-mercapto-ethanol derivatives have little or no activity. We conclude that the —SH group of CSU is very near the active site but is not required for catalytic activity and discuss possible alternative functions for this group. We also discuss the general use of enzyme-thionitro-benzoate mixed disulfides as intermediates in the preparation of specific derivatives of enzyme —SH groups and the potential use of enzyme thiocyanates, prepared through the thionitrobenzoate disulfides, in specific chemical cleavage of polypeptide chains at cysteine and cystine residues.