Investigation of the disordered loop of the α subunit of bacterial luciferase /
Bacterial luciferase is a 76 kD, heterodimeric protein comprised of homologous subunits. The enzyme catalyzes the conversion of FMNH₂, a long-chain aldehyde and molecular oxygen, to FMN, H₂O and the corresponding carboxylic acid with the emission of a photon of blue-green light. The light-emitting...
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
2001.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=725912311&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Bacterial luciferase is a 76 kD, heterodimeric protein comprised of homologous subunits. The enzyme catalyzes the conversion of FMNH₂, a long-chain aldehyde and molecular oxygen, to FMN, H₂O and the corresponding carboxylic acid with the emission of a photon of blue-green light. The light-emitting species is an excited flavin molecule that must be protected from solvent. Exposure of this species to solvent would lead to quenching of the excited state, resulting in a dark reaction. This protection was thought to be provided by a loop that is disordered in the crystal structure but had previously been shown to become ordered upon substrate binding. Genetic deletion of this loop resulted in an α subunit that was very similar to the β subunit in terms of length and structure. Biophysical techniques showed that the α[]₂₆₂-₂₉₀β heterodimer did not have any gross structural changes, although it did display a measurable subunit dissociation constant, unlike the wild-type enzyme. The ability of α[]₂₆₂β-₂₉₀β to produce light was reduced two orders of magnitude as compared with wild type. α[]₂₆₂-₂₉₀β retains the ability to bind substrate with nearly the same affinity as the wild-type enzyme. A method was developed to monitor the carboxylic acid produced by α[]₂₆₂-₂₉₀β to determine the mutant's ability to complete the chemistry of the bioluminescence reaction. The mutant is able to successfully accomplish the chemistry, although slightly less efficiently than the wild-type enzyme. These results indicate that the loop closes over the active site, protecting the high-energy intermediate from solvent, and allowing for a high quantum yield reaction. To further investigate the loop region, four mutants were made in which one quarter of the loop was substituted with alanines. These mutants were able to fold properly and assembled into stable heterodimers that did not display a subunit dissociation constant. These mutants had reduced activity. Substitution of alanines in the first and second quarters of the loop resulted in enzymes whose light production was decreased by two orders of magnitude as compared with wild type. Substitution in the third and fourth quarters reduced light production by only one order of magnitude. |
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| Item Description: | Vita. "Major Subject: Biochemistry". |
| Physical Description: | xii, 117 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilm Inc. |
| Bibliography: | Includes bibliographical references (leaves 112-116). |