Bacillus cereus beta-lactamase.  Reaction with N-bromosuccinimide and the properties of the product.
The effect of N-bromosuccinimide on the enzymatic activity and the conformation of a Bacillus cereus beta-lactamase (penicillin amido-beta-lactamase EC 3.5.2.6) was studied. Incubation with 10 muM N-bromosuccinimide caused over 95% decrease of the enzymatic activity within 15 min. Spectrophotometric titration with N-bromosuccinimide showed that the reaction proceeded in two steps. The half-inactivated enzyme was prepared by the reaction with N-bromosuccinimide and its properties examined. Amino acid analysis showed that the half-inactivated enzyme contained one residue of tryptophan less while other amino acid contents were similar. Neither the molecular weight nor the mobility in disc electrophoresis was changed. However, the affinity to a cephalexin-CH-Sepharose column was increased, and the Km value for cloxacillin was one-third that of the native enzyme, although that for benzylpenicillin was similar. These results indicate that a tryptophan residue sensitive to N-bromosuccinimide is essential for the maintenance of the rigid conformation and that its oxidation alters the enzyme in a manner such that a substrate with a bulky group in its side chain can form an enzyme-substrate complex more easily. In the native enzyme, the value of (f(a))(eff) (Lehrer, S.S. (1971) Biochemistry 10, 3254-3263), did not vary significantly in the absence or the presence of cloxacillin. In contrast, in the half-inactivated enzyme the presence of cloxacillin affected the conformation such that over two thirds of the tryptophyl fluorescence were accessible for quenching by KI, although about half was accessible in the absence of cloxacillin.
D001409:Bacillus cereus / Q000201:enzymology*; D001665:Binding Sites; D001974:Bromosuccinimide / Q000494:pharmacology; D002846:Chromatography, Affinity; D003023:Cloxacillin; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007461:Iodoacetates / Q000494:pharmacology; D007700:Kinetics; D010405:Penicillinase / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013388:Succinimides; D013997:Time Factors
D004791:Enzyme Inhibitors; D007461:Iodoacetates; D013388:Succinimides; D010405:Penicillinase; D001974:Bromosuccinimide; D003023:Cloxacillin