Carbodiimide-reactive carboxyl groups at the active site of an insect midgut trehalase

Carbodiimide modification of the Rhynchosciara americana midgut trehalase (alpha, alpha-trehalose glucohydrolase, EC 3.2.1.28) at different pH values revealed the existence of two essential groups (pKa 5.28 and pKa 7.74) for the trehalos activity. Those groups must be carboxyl groups since the alternative possibilities (sulfhydryl and phenol groups) have been discarded by selective modification and attempts to reactivate the modified enzyme with hydroxylamine. Furthermore, the increase of the pKa values of carbodiimide-reactive groups in the presence of dioxane supports further evidence that they are carboxyls. The results suggest the pKa 5.28 carboxyl is in the active site, while the pKa 7.74 carboxyl is in its neighborhood buried in the enzyme molecule. The possible role for the carbodiimide-reactive carboxyl groups in catalysis is discussed.