The use of mutant enzymes to probe the structure, function and folding pathway of bacterial luciferase /
Bacterial luciferase is a 76 kD, heterodimeric protein
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| Format: | Thesis Book |
| Language: | English |
| Published: |
[Place of publication not identified] :
[publisher not identified] ;
1997.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=736824831&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Bacterial luciferase is a 76 kD, heterodimeric protein comprised of homologous but not identical subunits. It catalyzes the conversion of FMNH2, 02 and a long chain aldehyde to FMN, H20 and the corresponding fatty acid with the emission of a photon of blue-green light. Amino acid substitution of []Asp113 with asparigine leads to an alteration of enzymatic kinetics, a 100-fold decrease in flavin binding affinity, drastically diminished bioluminescence, and a red shift of emitted light. The mutation also brings about significant changes in the enzyme's structure as shown by tryptophanyl fluorescence, circular dichroism, and limited proteolysis. Perturbations in the active center were detected by spectral changes in the bound flavin. It is proposed that the substitution of an asparagine for an aspartic acid at position []113 disrupts a hydrogen bond network which is essential to the structure of the flavin binding site of the enzyme. Two luciferase mutants, []D113N and JS16 (amino acids 159-162 are deleted from the [] subunit), were used in subunit exchange experiments where the formation of wild-type luciferase was detected by activity assay. A detailed mechanism for the folding and assembly of luciferase has been developed in our lab. These experiments were used to test the validity of the model for luciferase folding. All equilibrium and kinetic folding/unfolding experiments were carried out at 18 []C, pH 7.0 in 50 mM phosphate, and 0.5 mM DTT. Circular dichroism was used to identify the absolute configuration of the peroxyflavin of luciferase intermediate II as the 4a(R) isomer of 4a-hydroperoxyFMN. It has previously been demonstrated that only one of the 4a(R/S) stereoisomers of 4a-propylFMN is able to bind flavodoxin with substantial affinity. However the absolute stereochemistry of these 4a- propylflavins was not determined [Scola-Nagelschneider, et al. (1976) Eur. J. Biochem. 69, 305-314]. Consequently, a prediction of 4a-propylflavin stereochemistry is reported herein based on molecular modeling of 4a-propylFMN in the flavodoxin binding site. The stereochemical assignment of intermediate II was made by analogy of its CD spectrum to the CD spectrum of 4a(S)-propylFMN reported by Scola- Nagelschneider et al. (1976). |
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| Item Description: | Vita. "Major Subject: Chemistry". |
| Physical Description: | xiii, 116 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references: pages 103-114. |