Contribution of Lys276 to the conformational flexibility of the active site of glutamate decarboxylase from Escherichia coli.
Glutamate decarboxylase is a pyridoxal 5'-phosphate-dependent enzyme responsible for the irreversible alpha-decarboxylation of glutamate to yield 4-aminobutyrate. In Escherichia coli, as well as in other pathogenic and nonpathogenic enteric bacteria, this enzyme is a structural component of the glutamate-based acid resistance system responsible for cell survival in extremely acidic conditions (pH < 2.5). The contribution of the active-site lysine residue (Lys276) to the catalytic mechanism of E. coli 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, 400-fold less than that of the reaction between glutamate and pyridoxal 5'-phosphate 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.
Tramonti
Angela
A
Dipartimento di Scienze Biochimiche 'A. Rossi Fanelli' and Centro di Studio sulla Biologia Molecolare del CNR, Rome, Italy. debiase@caspur.it
John
Robert A
RA
Bossa
Francesco
F
De Biase
Daniela
D
D001665:Binding Sites; D002942:Circular Dichroism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005968:Glutamate Decarboxylase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D008239:Lysine; D009154:Mutation; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D029968:Escherichia coli Proteins; D018698:Glutamic Acid; D011732:Pyridoxal Phosphate; D005968:Glutamate Decarboxylase; D008239:Lysine