Mechanism of the Inactivation of Guinea Pig Liver Transglutaminase by Tetrathionate

Abstract

Abstract Treatment of transglutaminase with 2 moles of sodium tetrathionate results in almost complete losses in the transferase and hydrolysis activities of the enzyme toward the substrate, benzyloxycarbonyl-l-glutaminylglycine. The calcium-dependent esterase activity of transglutaminase toward p-nitrophenyl acetate is only partially lost as a result of this treatment. A change in the mechanism of calcium activation, however, is evident from the relationship of the velocities of ester hydrolysis to metal ion concentrations. The losses in the catalytic activities of transglutaminase are accompanied by a concomitant disappearance of four sulfhydryl groups in the enzyme protein. Enzyme inactivated with 35S-labeled tetrathionate showed incorporation of only small amounts of isotope, equivalent to 0.1 to 0.15 mole per mole of enzyme protein. This is in accord with evidence that the loss of the four —SH groups is the result of formation of two intramolecular disulfide bridges. 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 bridges formed by tetrathionate treatment. Brief incubation of the modified enzyme with dithiothreitol reduces one of these two disulfide bonds as evidenced by the recovery of two —SH groups. No restoration of enzymatic activities, however, accompanies this disulfide cleavage. Upon continued incubation with dithiothreitol, the remaining two —SH groups are recovered together with a parallel restoration of all enzymatic activities. It would appear that the catalytic changes which occur as a result of tetrathionate treatment are associated with formation of a single intramolecular disulfide bridge in the enzyme protein.