Jan 13, 1995

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

The Journal of Biological Chemistry
J OsunaX Soberón

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 muta...Continue Reading

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Mentioned in this Paper

Asparagine
Aspartic Acid, Magnesium-Potassium (2:1:2) Salt
Beta-Lactamase
Oligonucleotide Primers
Catalysis
Hydrogen-Ion Concentration
Mutagenesis Process

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