[TEM-type beta-lactamase coded by the plasmid from Citrobacter sp].
beta-Lactamase was isolated from the cells of E. coli, strain 1039, a transconjugant carrying the plasmid first detected in Citrobacter sp. beta-Lactamase was purified to obtain a homogeneous preparation. The activity of the enzyme was estimated by a modification of the potentiometric method providing determination of up to 0.1 mumol of penicilloinic acid. The activity of the pure enzyme was 206 mumol per 1 mg of protein per minute. The molecular weight determined by gel filtration was 21500. The substrate profile of beta-lactamase corresponded to the TEM type. Preliminary incubation with methicillin resulted in a significant decrease in the enzyme activity. Carbenicillin, dicloxacillin, oxacillin and cephalothin induced no decrease in the activity of the enzyme when subjected to preliminary incubation with it. The use of the functional group reagents allowed detecting residues of serine and lysine in the enzyme active centre or close to it. Km and Kcat for some antibiotics were evaluated. The ratio of alpha- and beta-structures in the molecule of beta-lactamase was determined with the method of circular dichroism (CD). The fraction of the alpha-spiral areas amounted to 30 +/- 5 per cent and the fraction of beta-structures amounted to 20 +/- 5 per cent. On attachment of methicillin to the molecule of the enzyme the ratio of alpha- and beta-structures did not change which may be considered as a preliminary indication of the centre "immobility" in the process of catalysis. Slow hydroysis of methicillin by beta-lactamase was shown with the method of CD.
D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002954:Citrobacter / Q000201:enzymology / Q000235:genetics*; D003227:Conjugation, Genetic; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005815:Genetic Code; D007700:Kinetics; D010957:Plasmids; D001618:beta-Lactamases / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification
D001618:beta-Lactamases
beta-laktamaza TEM-tipa, kodiruemaia plazmidoĭ iz Citrobacter sp.