The Construction and Kinetic Analysis of Specific Mutants of β-Lactamase

Citation

Carroll, Steven S. (1987) The Construction and Kinetic Analysis of Specific Mutants of β-Lactamase. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/2mg4-x771. https://resolver.caltech.edu/CaltechTHESIS:08302019-102809124

Abstract

Mutants of RTEM-1 β-lactamase, an enzyme responsible for bacterial resistance to β-lactam antibiotics, have been constructed by cassette mutagenesis. β-Lactamases have been classified into three groups based on catalytic and structural similarities. Class A B-lactamases, of which the RTEM-1 enzyme is a member, maintain a conserved triad of amino acids about the active site serine, Ser-Thr-X-Lys, where X is not conserved. Endonuclease restriction sites flanking the codons of the active site residues were used to remove the portion of the β-lactamase gene coding for the wild-type active site sequence. A duplex strand of synthetic oligonucleotides spanning the removed region of the gene and coding for the desired mutations was ligated in place of the wild-type sequence. Subsequent chemical sequencing of the mutated plasmids by the Maxam-Gilbert technique confirmed the presence of the mutations.

A mutant converting Thr71 to cysteine, designated Thr71 → Cys, was constructed and shown to impart to cells a phenotype resistant to high levels of ampicillin (>350 mg/L). Purification of the mutant to homogeneity was accomplished using plasmid pJN which contains the β-lactamase gene under control of the tac promoter and the gene for kanamycin resistance. Cells were grown at 37°C and induction was carried out at 30°C to reduce protein losses due to the thermal instability of the mutant. The Thr71 → Cys mutant retains 12% of the efficiency of the wild-type enzyme on benzylpenicillin 3% on cephalothin, and 9% on 6-aminopenicillanic acid. The mutant has an altered pH activity profile, with activity declining more rapidly at alkaline pH than for the wild-type enzyme. The mutant is susceptible to inactivation by the sulfhydryl reagent pCMB under conditions where the activity of the wild-type enzyme is not affected. The mutant is inhibited competitively by borate ion. The Thr71 → Cys mutant is less stable to thermal denaturation than the wildtype enzyme, as are all other mutants at position 71.

The conserved lysine at position 73 of β-lactamase was replaced with all other amino acids and an amber stop codon by cassette mutagenesis using synthetic oligonucleotides containing a mixture of bases in place of the lysine codon. After ligation the mixtures of plasmids were used to transform E. coli to tetracycline resistance. Plasmids derived from one hundred colonies were sequenced by the Maxam-Gilbert method. Because of an uneven mixture of bases in the mixed codon not all twenty amino acids were located in the first one hundred colonies. Mutants Lys73 → Asp, Glu and Pro were constructed separately using the same three fragment ligation scheme. None of the mutants at position 73 were able to confer to cells a phenotype resistant to even low levels of ampicillin. Western blot analysis of whole cell extracts revealed mutants with all nineteen amino acid replacements present in amounts not significantly different from the amount of the wild-type enzyme.

Purification of mutants Lys73 → Arg and Cys was accomplished with the use of plasmid pJN and the tac promoter. Kinetic analysis revealed both mutants maintain 0.01% of the activity of the wild-type enzyme on benzylpenicillin. The Michaelis constant, K_M, was not significantly different from the value of the wild-type. The Lys73 → Cys mutant was slightly less stable to thermal denaturation than the wildtype enzyme. The pH-activity profile of the. Lys73 → Cys mutant showed a maximum at pH 8.3 while the wild-type activity is maximal at pH 6.5. Treatment of the Lys73 → Cys mutant with ethylenimine, which converts cysteine to aminoethylcysteine, under mildly denaturing conditions resulted in a dramatic increase in specific activity of the mutant. Purification of the derivatized mutant by anion exchange on FPLC and kinetic analysis showed the derivatized mutant regained 62% of the catalytic efficiency of the wild-type enzyme.

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