Đang chuẩn bị nút TẢI XUỐNG, xin hãy chờ
Tải xuống
Glutamate decarboxylase is a pyridoxal 5¢-phosphate-dependent enzyme responsible for the irreversiblea-decar-boxylation of glutamate to yield 4-aminobutyrate. In Escherichia coli, as well as in other pathogenic and non-pathogenic enteric bacteria, this enzyme is a structural component of the glutamate-based acid resistance system responsible for cell survival in extremely acidic conditions (pH | Eur. J. Biochem. 269 4913-4920 2002 FEBS 2002 doi 10.1046 j.1432-1033.2002.03149.x Contribution of Lys276 to the conformational flexibility of the active site of glutamate decarboxylase from Escherichia coli Angela Tramonti1 Robert A. John2 Francesco Bossa1 and Daniela De Biase1 1 Dipartimento di Scienze Biochimiche A. Rossi Fanelli and Centro di Studio sulla Biologia Molecolare del CNR Rome Italy Cardiff School of Biosciences Cardiff UK Glutamate decarboxylase is a pyridoxal 5 -phosphate-dependent enzyme responsible for the irreversible a-decar-boxylation of glutamate to yield 4-aminobutyrate. In Escherichia coli as well as in other pathogenic and non-pathogenic 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 40 . -fokl less titan 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 th e protein less flexible and its active site less accessible to substrate and cofactor. Keywords glutamate decarboxylase pyridoxal 5 .
