Mechanism of the inactivation of guinea pig liver transglutaminase by 5,5'-dithiobis-(2-nitrobenzoic acid).

Reaction of 5,5’-dithiobis(24trobenzoic acid) (DTNB) with transglutaminase in the absence of calcium ion results in losses in the transferase and hydrolysis activities of the enzyme toward the substrate, benzyloxycarbonyl-L-glutaminylglycine (Z-L-glutaminylglycine). These activities are reduced 70 to 100% by reaction with 1 to 1.5 eq of DTNB. The calcium-dependent esterase activity of transglutaminase toward p-nitrophenyl acetate is not lost by this treatment. However, the activator constant of Ca++ for this esterase activity, 1.7 X low3 M at pH 7, is several fold higher than that observed for the esterase activity of native enzyme. The esterase activity of DTNB-modified enzyme is not inhibited by Z-L-glutaminylglycine. This is in contrast to the effective inhibition aEorded by this substrate toward the esterase activity of native transglutaminase. Z-L-Glutaminylglycine protects the enzyme against inactivation by DTNB. This protection is observed only in the presence of Ca++, which is essential for the binding of this substrate. Complete loss in transferase and hydrolysis activities is accompanied by the loss of 2 sulfhydryl residues in the enzyme and the concomitant release of 2 eq of 5-thio-2nitrobenzoic acid (or its thioquinone) for each equivalent of DTNB used for the modification. The loss in -SH groups appears to be the result of formation of a single intramolecular disulfide bridge in the enzyme. The DTNB-inactivated enzyme is readily reactivated by treatment with dithiothreitol. Peptide mapping studies show that a single -SH group of transglutaminase previously identified as essential for all catalytic activities is not a component of the disulfide bridge formed as a result of DTNB treatment. These tindings suggest that the changes that occur in transglutaminase upon treatment with DTNB in the absence of Ca+f result in a loss in binding properties for glutamine substrate. In contrast to the inactivation of transglutaminase by low levels of DTNB in the absence of Ca+f, inactivation of the enzyme at a high Caf+ concentration (50 mM) requires several fold higher DTNB levels. Furthermore, DTNB treatment at high Ca++ levels results in parallel losses in all three catalytic activities of the enzyme. This finding is in accord with earlier observations that a Ca++-induced conformational change in transglutaminase affects the reactivity of the enzyme protein toward modifying agents. Previous studies have shown that guinea pig liver transglutaminase contains 17 or 18 sulfhydryl groups per molecule and is devoid of disuhide bonds (1, 2). A C&+-catalyzed inactivation of this enzyme has been correlated with the loss of 4 -SH groups, the probable result of the formation of two intramolecular disulfide bonds (2). The divalent cation, calcium, that is essential for transglutaminase activity (3, 4) is required for this inactivation. Treatment of the inactive disulfide form of the enzyme with dithiothreitol results in a rapid, full reactivation accompanied by restoration of SH groups to the level of the native enzyme. The -SH group of a single cysteine residue, previously identified as essential for transglutaminase activity (1, 5), is apparently not a component of the disulfide bonds formed by Cu++ treatment. In a further effort to relate the -SH groups of transglutaminase to its catalytic properties, advantage was taken of an earlier observation that treatment of the enzyme with low levels of 5,5’-dithiobis(2-nitrobenzoic acid) resulted in losses in enzymatic activity. A preliminary study with 35S-labeled DTNBl showed that, although 70 to 90% of the transferase activity of the enzyme was lost upon incubation with 1 eq of this reagent in the absence of Ca++, no 3%-containing material was irreversibly bound to the enzyme protein.2 The experiments reported here supply evidence that the enzymatic changes that occur upon treatment of transglutaminase with 1 eq of DTNB in the absence of Ca++ are the result of the formation of a single intramolecular disulfide bond.