Article (Scientific journals)
Role of the Conserved Amino Acids of the 'Sdn' Loop (Ser130, Asp131 and Asn132) in a Class a Beta-Lactamase Studied by Site-Directed Mutagenesis
Jacob, Françoise; Joris, Bernard; Lepage, Sophie et al.
1990In Biochemical Journal, 271 (2), p. 399-406
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Abstract :
[en] Ser130, Asp131 and Asn132 ('SDN') are highly conserved residues in class A beta-lactamases forming one wall of the active-site cavity. All three residues of the SDN loop in Streptomyces albus G beta-lactamase were modified by site-directed mutagenesis. The mutant proteins were expressed in Streptomyces lividans, purified from culture supernatants and their kinetic parameters were determined for several substrates. Ser130 was substituted by Asn, Ala and Gly. The first modification yielded an almost totally inactive protein, whereas the smaller-side-chain mutants (A and G) retained some activity, but were less stable than the wild-type enzyme. Ser130 might thus be involved in maintaining the structure of the active-site cavity. Mutations of Asp131 into Glu and Gly proved to be highly detrimental to enzyme stability, reflecting significant structural perturbations. Mutation of Asn132 into Ala resulted in a dramatically decreased enzymic activity (more than 100-fold) especially toward cephalosporin substrates, kcat. being the most affected parameter, which would indicate a role of Asn132 in transition-state stabilization rather than in ground-state binding. Comparison of the N132A and the previously described N132S mutant enzymes underline the importance of an H-bond-forming residue at position 132 for the catalytic process.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Jacob, Françoise
Joris, Bernard ;  Université de Liège - ULiège > Centre d'ingénierie des protéines
Lepage, Sophie
Dusart, Jean
Frère, Jean-Marie ;  Université de Liège - ULiège > Département des sciences de la vie > Département des sciences de la vie
Language :
English
Title :
Role of the Conserved Amino Acids of the 'Sdn' Loop (Ser130, Asp131 and Asn132) in a Class a Beta-Lactamase Studied by Site-Directed Mutagenesis
Publication date :
15 October 1990
Journal title :
Biochemical Journal
ISSN :
0264-6021
eISSN :
1470-8728
Publisher :
Portland Press, United Kingdom
Volume :
271
Issue :
2
Pages :
399-406
Peer reviewed :
Peer Reviewed verified by ORBi
Available on ORBi :
since 02 January 2012

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