Site-directed mutagenesis and substrate-induced inactivation of beta-lactamase I.
The substrate-induced inactivation of beta-lactamase I from Bacillus cereus 569/H has been studied. Both the wild-type enzyme and mutants have been used. The kinetics follow a branched pathway of the type recently analysed [Waley (1991) Biochem. J. 279, 87-94]. The substrate cloxacillin (a penicillin) formed an acyl-enzyme (characterized by m.s.), and it was probably the instability of this intermediate that brought about inactivation. A disulphide bond was introduced into beta-lactamase I (the wild-type enzyme lacks this bond) by site-directed mutagenesis: Ala-77 and Ala-123 were replaced by cysteine. Spontaneous oxidation yielded the disulphide. The activity of this newly cross-linked enzyme was a little diminished, but the stability towards inactivation by cloxacillin was not increased. A second mutant of beta-lactamase I was studied: this mutant lacked the first 17 residues, i.e. the first alpha-helix. The mutant had reduced activity towards ordinary (non-inactivating) substrates and no hydrolysis of cloxacillin could be detected. These mutant enzymes were expressed in Bacillus subtilis, and were purified from the extracellular medium.
Thornewell
S J
SJ
University of Oxford, Sir William Dunn School of Pathology, U.K.
Waley
S G
SG
D002511:Cephalosporins / Q000378:metabolism / Q000494:pharmacology; D003023:Cloxacillin / Q000378:metabolism / Q000494:pharmacology*; D004355:Drug Stability; D004789:Enzyme Activation / Q000187:drug effects; D006868:Hydrolysis; D007700:Kinetics; D016297:Mutagenesis, Site-Directed / Q000235:genetics / Q000502:physiology*; D010400:Penicillin G / Q000378:metabolism / Q000494:pharmacology; D010405:Penicillinase / Q000235:genetics* / Q000378:metabolism*
D002511:Cephalosporins; C025163:cephalosporin C; D010405:Penicillinase; C021720:nitrocefin; D003023:Cloxacillin; D010400:Penicillin G