Mechanism of the Inactivation of Guinea Pig Liver Transglutaminase by 5,5'-Dithiobis-(2-nitrobenzoic Acid)
John M. Connellan 1 and J. E. Folk 1
From the
1 From the National Institutes of Health, National Institute of Dental Research, Enzyme Chemistry Section, Laboratory of Biochemistry, Bethesda, Maryland 20014
Reaction of 5,5'-dithiobis(2-nitrobenzoic 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 10-3 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 afforded 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-2-nitrobenzoic 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 findings suggest that the changes that occur in transglutaminase upon treatment with DTNB in the absence of Ca++ 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++, inactivation of the enzyme at a high Ca++ 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.
Submitted on February 10, 1969
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