title: Substitution of Asp for Asn at position 132 in the active site of TEM beta-lactamase. Activity toward different substrates and effects of neighboring residues.
abstract: Using a random, combinatorial scheme of mutagenesis directed against the conserved SDN region of TEM beta-lactamase, and selective screening in ampicillin-plates, we obtained the N132D mutant enzyme. The kinetic characterization of this mutant indicated relatively small effects compared to the wild-type. Both pK1 and pK2 for catalysis were decreased about 1 unit relative to the pK's for the wild type. This effect was predominantly due to changes in Km. In contrast to the wild-type, the pH-rate profiles of the mutant showed that Km for several side chain-containing penicillin substrates increases when the pH is above 5.5. 6-Aminopenicillanic acid, which lacks a side chain, did not show this effect. With benzylpenicillin, ampicillin, and carbenicillin, kcat for the mutant showed a similar pH dependence as the wild type. With 6-aminopenicillanic acid, kcat for the mutant was greater than that for the wild type. The nature of the 104 side chain may affect the environment of Asp132; double mutants N132D/E104X (where X can be Q or N) are unable to confer antibiotic resistance to bacterial cells. The computed contact interactions from modeling substrate complexes between benzylpenicillin or 6-aminopenicillanic acid with the N132D mutant confirmed the importance of the protonation state of residue Asp132 for the complex stability with side chain-containing substrates. The data indicate that the contact between the side chain of residue 132 and the substrate is relevant for the ground state recognition, but because of close contact with several important groups in its neighborhood, residue 132 is also indirectly involved in the catalytic step of the wild-type enzyme.
authors: Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca.
authors: J
authors: J
authors: Osuna
authors: H
authors: H
authors: Viadiu
authors: A L
authors: AL
authors: Fink
authors: X
authors: X
authors: Soberón
mesh_terms: D001216:Asparagine / Q000378:metabolism*; D001224:Aspartic Acid / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D017931:DNA Primers; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016296:Mutagenesis; D001618:beta-Lactamases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D001224:Aspartic Acid; D001216:Asparagine; D001618:beta-Lactamases