Abstract
Abstract Guinea pig liver transglutaminase is rapidly and progressively inactivated during incubation with copper salts in the presence of calcium, a divalent cation essential for enzymatic activity. Substrate exerts some protection against this inactivation. Complete loss of transglutaminase activity is accompanied by a concomitant loss of approximately 4 sulfhydryl residues. This loss of enzyme sulfhydryl groups appears to be the result of formation of 2 intramolecular disulfide bridges. The Cu++-inactivated enzyme is readily reactivated by treatment with dithiothreitol or potassium cyanide. Peptide mapping experiments show that a sulfhydryl group of transglutaminase previously identified as essential for catalytic activity is not a component of the disulfide bonds formed as a result of Cu++ treatment. The dissociation constant (Kd) for calcium (7.1 ± 1.0 x 10-3 m) estimated from the rate of inactivation of transglutaminase by Cu++ as a function of calcium ion concentration is in close agreement with Kd values for calcium determined by other direct binding experiments. It is suggested, on the basis of earlier findings, that a calcium-induced conformational change in the enzyme protein is essential for Cu++-catalyzed formation of an inactive disulfide form of transglutaminase.
Highlights
Guinea pig liver transglutaminase is rapidly and progressively inactivated during incubation with copper salts in the presence of calcium, a divalent cation essential for enzymatic activity
The studies reported in this communication were stimulated by an observation that concomitant losses in transglutaminase -SH groups and catalytic activity occur as a result of incubation of this enzyme with low levels of copper only in the presence of calcium, an ion essential for enzymatic activity.’
The results support the premise that complete loss in catalytic activity is associated with the formation of 2 disulfide bonds in the enzyme protein and that the sulfhydryl groups from which these covalent bonds are formed are not essential per se for enzymatic activity
Summary
Guinea pig liver transglutaminase is rapidly and progressively inactivated during incubation with copper salts in the presence of calcium, a divalent cation essential for enzymatic activity. The dissociation constant (KD) for calcium (7.1 + 1.0 X 10U3 M) estimated from the rate of inactivation of transglutaminase by Cuff as a function of calcium ion concentration is in close agreement with KD values for calcium determined by other direct binding experiments It is suggested, on the basis of earlier findings, that a calcium-induced conformational change in the enzyme protein is essential for Cu++-catalyzed formation of an inactive disulfide form of transglutaminase. The studies reported in this communication were stimulated by an observation that concomitant losses in transglutaminase -SH groups and catalytic activity occur as a result of incubation of this enzyme with low levels of copper only in the presence of calcium, an ion essential for enzymatic activity.’. The studies reported in this communication were stimulated by an observation that concomitant losses in transglutaminase -SH groups and catalytic activity occur as a result of incubation of this enzyme with low levels of copper only in the presence of calcium, an ion essential for enzymatic activity.’ The results support the premise that complete loss in catalytic activity is associated with the formation of 2 disulfide bonds in the enzyme protein and that the sulfhydryl groups from which these covalent bonds are formed are not essential per se for enzymatic activity
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