Site-directed mutagenesis of beta-lactamase I: role of Glu-166.
Two Glu-166 mutants of beta-lactamase I from Bacillus cereus 569/H were constructed: one with a lengthened side chain (E166Cmc, the S-carboxymethylcysteine mutant) and the other with the side chain shortened and made non-polar (E166A). Their kinetic properties were studied and compared with those of...
| Main Authors: | , , , |
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| Format: | Journal article |
| Language: | English |
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1994
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| _version_ | 1826264259818946560 |
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| author | Leung, Y Robinson, C Aplin, RT Waley, S |
| author_facet | Leung, Y Robinson, C Aplin, RT Waley, S |
| author_sort | Leung, Y |
| collection | OXFORD |
| description | Two Glu-166 mutants of beta-lactamase I from Bacillus cereus 569/H were constructed: one with a lengthened side chain (E166Cmc, the S-carboxymethylcysteine mutant) and the other with the side chain shortened and made non-polar (E166A). Their kinetic properties were studied and compared with those of the wild-type and the E166D mutant (with a shortened side chain) previously made by Gibson, Christensen and Waley (1990) (Biochem. J. 272, 613-619). Surprisingly, with good penicillin substrates, Km, kcat. and kcat./Km of the two conservative mutants (E166Cmc and E166D) are similar to those of the non-conservative mutant E166A. Their kcat. values are 3000-fold lower than that of the wild-type enzyme, showing that Glu-166 is a very important residue. The acylenzyme intermediate of E166A and a good substrate, penicillin V, was trapped by acid-quench and observed by electrospray ionization mass spectrometry, suggesting that Glu-166 is more important in catalysing the deacylation step than the acylation step. The beta-lactamase I E166A mutant is about 200-fold more active than the Bacillus licheniformis E166A mutant with nitrocefin or 6 beta-furylacryloyl-amidopenicillanic acid as substrate. This suggested that other groups in the active site of the beta-lactamase I mutant may activate the catalytic water molecule for deacylation. |
| first_indexed | 2024-03-06T20:04:53Z |
| format | Journal article |
| id | oxford-uuid:289539b1-e4f9-4396-82e5-c6a80617ded8 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T20:04:53Z |
| publishDate | 1994 |
| record_format | dspace |
| spelling | oxford-uuid:289539b1-e4f9-4396-82e5-c6a80617ded82022-03-26T12:13:44ZSite-directed mutagenesis of beta-lactamase I: role of Glu-166.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:289539b1-e4f9-4396-82e5-c6a80617ded8EnglishSymplectic Elements at Oxford1994Leung, YRobinson, CAplin, RTWaley, STwo Glu-166 mutants of beta-lactamase I from Bacillus cereus 569/H were constructed: one with a lengthened side chain (E166Cmc, the S-carboxymethylcysteine mutant) and the other with the side chain shortened and made non-polar (E166A). Their kinetic properties were studied and compared with those of the wild-type and the E166D mutant (with a shortened side chain) previously made by Gibson, Christensen and Waley (1990) (Biochem. J. 272, 613-619). Surprisingly, with good penicillin substrates, Km, kcat. and kcat./Km of the two conservative mutants (E166Cmc and E166D) are similar to those of the non-conservative mutant E166A. Their kcat. values are 3000-fold lower than that of the wild-type enzyme, showing that Glu-166 is a very important residue. The acylenzyme intermediate of E166A and a good substrate, penicillin V, was trapped by acid-quench and observed by electrospray ionization mass spectrometry, suggesting that Glu-166 is more important in catalysing the deacylation step than the acylation step. The beta-lactamase I E166A mutant is about 200-fold more active than the Bacillus licheniformis E166A mutant with nitrocefin or 6 beta-furylacryloyl-amidopenicillanic acid as substrate. This suggested that other groups in the active site of the beta-lactamase I mutant may activate the catalytic water molecule for deacylation. |
| spellingShingle | Leung, Y Robinson, C Aplin, RT Waley, S Site-directed mutagenesis of beta-lactamase I: role of Glu-166. |
| title | Site-directed mutagenesis of beta-lactamase I: role of Glu-166. |
| title_full | Site-directed mutagenesis of beta-lactamase I: role of Glu-166. |
| title_fullStr | Site-directed mutagenesis of beta-lactamase I: role of Glu-166. |
| title_full_unstemmed | Site-directed mutagenesis of beta-lactamase I: role of Glu-166. |
| title_short | Site-directed mutagenesis of beta-lactamase I: role of Glu-166. |
| title_sort | site directed mutagenesis of beta lactamase i role of glu 166 |
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