Contribution of Lys276 to the conformational flexibility of the active site of glutamate decarboxylase from Escherichia coli
2002
Tramonti, Angela | John, Robert A. | Bossa, Francesco | De Biase, Daniela
Glutamate decarboxylase is a pyridoxal 5′âphosphateâdependent enzyme responsible for the irreversible αâdecarboxylation of glutamate to yield 4âaminobutyrate. In Escherichia coli, as well as in other pathogenic and nonpathogenic enteric bacteria, this enzyme is a structural component of the glutamateâbased acid resistance system responsible for cell survival in extremely acidic conditions (pHâ<â2.5). The contribution of the activeâsite lysine residue (Lys276) to the catalytic mechanism of E.âcoli glutamate decarboxylase has been determined. Mutation of Lys276 into alanine or histidine causes alterations in the conformational properties of the protein, which becomes less flexible and more stable. The purified mutants contain very little (K276A) or no (K276H) cofactor at all. However, apoenzyme preparations can be reconstituted with a full complement of coenzyme, which binds tightly but slowly. The observed spectral changes suggest that the cofactor is present at the active site in its hydrated form. Binding of glutamate, as detected by external aldimine formation, occurs at a very slow rate, 400âfold less than that of the reaction between glutamate and pyridoxal 5′âphosphate in solution. Both Lys276 mutants are unable to decarboxylate the substrate, thus preventing detailed investigation of the role of this residue on the catalytic mechanism. Several lines of evidence show that mutation of Lys276 makes the protein less flexible and its active site less accessible to substrate and cofactor.
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