Val-237 for Ala substitution in the TEM-2 β-lactamase dramatically alters the catalytic efficiencies towards carbenicillin and ticarcillin |
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Authors: | Michel Barthé lé my,Jean Pé duzzi,David Rowlands,Gé rard Paul,Gilles Moreau,Roger Labia |
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Affiliation: | Muséum National Histoire Naturelle, CNRS URA 401, 63 rue Buffon, 75231 Paris Cedex 05, France; Centre de Recherche Roussel-Uclaf, 93230 Romainville, France; CHU Cochin, Laboratoire de Bactériologie, 75014 Paris, France |
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Abstract: | Abstract The mutant 554 of TEM-2 β-lactamase was selected for a decrease in the resistance to carbenicillin of an Escherichia coli K12 carrier. The amino acid sequence of the mutant β-lactamase was determined by manual Edman degradation analysis of proteolytic peptides. A single substitution Val for Ala was localized at position 237. The mutant exhibited only 2% of the catalytic efficiency of the wild-type enzyme towards carbenicillin and ticarcillin, whereas it retained 30–60% of the hydrolytic activity towards other penicillin and cephalosporin substrates. Carfecillin, the phenyl ester of the side-chain carboxyl group of carbenicillin, was hydrolysed as a good substrate. This suggests that the behaviour of the mutant enzyme towards carbenicillin may result from ionic rather than steric constraints. A molecular model of the Val-237 TEM-2 mutant suggests possible electrostatic interaction between Glu-171 and the carboxylic group of the side chain of carbenicillin. |
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Keywords: | β-Lactamase mutagenesis Amino acid sequence TEM-type molecular model |
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