Role of Residue Lys315 in the Mechanism of Action of the Enterobacter cloacae 908R .beta.-Lactamase
| Autor: | Deidre Cooke, Jean-Marie Frère, Didier Monnaie, Staffan Normark, Jacqueline Marchand-Brynaert, Alain Dubus |
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| Rok vydání: | 1994 |
| Předmět: |
Magnetic Resonance Spectroscopy
Spectrophotometry Infrared Stereochemistry Molecular Sequence Data Penicillins Biochemistry beta-Lactamases Substrate Specificity Acylation Residue (chemistry) chemistry.chemical_compound Tetrahedral carbonyl addition compound Enterobacter cloacae Escherichia coli Histidine Amino Acid Sequence Carboxylate Conserved Sequence chemistry.chemical_classification Base Sequence biology Chemistry Lysine Active site Hydrogen-Ion Concentration biology.organism_classification Recombinant Proteins Kinetics Enzyme Oligodeoxyribonucleotides Mutagenesis Site-Directed biology.protein Plasmids |
| Zdroj: | Biochemistry. 33:5193-5201 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi00183a024 |
| Popis: | The role of the highly conserved Lys315 residue in the catalytic mechanism of a class C beta-lactamase has been probed by site-directed mutagenesis. Lys315 has been replaced by a histidine in the Enterobacter cloacae 908R beta-lactamase, thus introducing a tritratable group to probe the role of the positive charge, and by a glutamine. The effects of these mutations have been studied on the kinetics of penicillin G and cephalothin turnover and on the pre-steady-state kinetics with carbenicillin at different pH. Results showed that substrate binding was not impaired by the mutations, so that an interaction with the substrate-free carboxylate in the Henri-Michaelis complex could be ruled out. Lys315 must have a catalytic role as shown by the decreased acylation and deacylation rates observed with the mutant enzymes. The mutants exhibited a lower activity at acidic pH, and this observation could be correlated with a decreased affinity for (3-aminophenyl)boronate, a compound devoid of free carboxylate which binds to the active site and forms an adduct mimicking the tetrahedral intermediate. This suggested that Lys315 was somehow involved in accelerating the nucleophilic substitutions along the reaction pathway. The study was extended to modified substrates where the free carboxylate had been esterified. Neither acylation nor deacylation seemed severely impaired with these compounds, showing that the interaction between the enzyme and the substrate-free carboxylate did not play a major role in catalysis. |
| Databáze: | OpenAIRE |
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